Template:Pitt/labnotebook2

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Latest revision as of 22:46, 17 October 2014

PCR Reaction of Hsp60 Promoter June 25, 2014

Purpose: Amplification of Hsp60 Promoter for insertion into biobrick

Reaction Reagents:

1. mCherry Fushion hsp60 promoter template

2. Primers

a. Hsp60BBfwd

b. Hsp60rev comps

c. RBS_BBrev comps

3. PCR Buffer

4. Double distilled water

5. DNA Polymerase

6. 1kb DNA Ladder

Primer Numbering and Concentration:

1. Hsp60BBfwd: 29.1 nmol/100ul

2. Hsp60rev comps: 26.2 nmol/100ul

3. RBS_BBrev comps 27.7 nmol/100ul

Final Concentration 100 uM of DNA

Reagent Table 1:

Reagent	3x (ul)
Clear Color Buffer	12
dNTP's	1.2
Primers: 1 + 3	6
Template DNA	3
DNA Polymerase	0.6
ddH₂O	37.2
Total Volume	60

Reagent Table 2:

Reagent	3x (ul)
Clear Color Buffer	12
dNTP's	1.2
Primers: 1 + 2	6
Template DNA	3
DNA Polymerase	0.6
ddH₂O	37.2
Total Volume	60

Loading Buffer Concentration Table:

x= total loading volume

Reaction loaded (ul)	Loading Buffer (ul)
4	1
5	1.25
8	2
10	2.5

Lane

Sample

Amount Loaded (ul)

1

1kb DNA Ladder

2

(1) Hsp60BBfwd

(3) RBS_BBrev

6.25

3

Control, no primers

6.25

4

Ladder

6.25

5

(1) Hsp60BBfwd

(2) Hsp60BBrev

6.25

6

Control, no primers

6.25

It seems that too much template DNA was used hence the bands did not travel far

Liquid Culture with DAM- E. coli in LB

Thursday, June 26, 2014

1. Set up 3 liquid cultures with 4 ml of LB each and 4 ul of Ampicillin (1:1000 Concentration)

2. Incubated in 37˚C shaker overnight

Mini-Prep DAM- Negative Liquid Culture

Friday, June 27, 2014

Buffer Preparation:

Resuspension Solution: add provided RNAse Al store at 4˚C for up to 6 months

Wash Solution: Add 35 ml of 96% EtOH to 20 ml wash solution. Store at room temperature (RT); we have 100% EtOH

1. Pipet 1.5 mL of culture into microcentrifuge tube and spin for 2 minutes

2. Resuspend pellet in 250 mL of Resuspension solution

3. Add 250 mL of Lysis solution and mix by inverting tube

4. Add 360 mL of neutralization and mix immediately by inversion 4-6 times

5. Centrifuge for 5 minutes to pellet cell debris

6. Transfer supernatant to spin column

7. Centrifuge for 1 minute and discard flow through

8. Add 500 mL of wash solution and centrifuge for 30-60 seconds and discard flow through

9. Repeat wash using 500 mL of wash solution

10. Transfer spin column to fresh 1.5 mL tubes and add 50 mL of elution buffer to center of column

11. Incubate at RT for 2 minutes centrifuge for 2 minutes

12. Store flow through (purified plasmid DNA) at -20 degree Celsius.

Result:

Tube	Concentration (ng/uL)
1	29.1
2	29.5
3	18.4
4	25.9
5	18.5
6	16.8
7	23.9
8	15.6

PCR of Hsp60 with and without RBS

Friday, June 27, 2014

Making a 1:13 dilution of our template DNA which is 140 ng/uL

Components	Amount (ul)
deionized water	12
Template DNA	1

Reagent Table 1 (w/o RBS):

Reagent	3x (ul)
Clear Color Buffer	12
dNTP's	1.2
Primers: 1 + 3	3
Template DNA	3
DNA Polymerase	0.6
ddH₂O	37.2
Total Volume	57

Reagent Table 2 (w/ RBS):

Reagent	3x (ul)
Clear Color Buffer	12
dNTP's	1.2
Primers: 1 + 2	3
Template DNA	3
DNA Polymerase	0.6
ddH₂O	37.2
Total Volume	57

Primer Numbering and Concentration:

1. Hsp60BBfwd: 29.1 nmol/100ul

2. Hsp60rev comps: 26.2 nmol/100ul

3. RBS_BBrev comps 27.7 nmol/100ul

Final Concentration 100 uM of DNA

1.25 uL of loading dye + 5 uL PCR reaction

2ul 1 kb DNA ladder

Started gel at 4:26 pm

Stopped gel at 4:55 pm

Results:

There were no PCR Products

PCR with Hsp60

Monday, June 30, 2014

Reagent Table 1 (w/o RBS):

Reagent	2.5x (ul)
Clear Color Buffer	25
dNTP's	2.5
Primers: 1 + 3	12 (6 each)
Template DNA	6
Phusion	1.25
ddH₂O	77.5
Total Volume	125

Reagent Table 2 (w/ RBS):

Reagent	2.5x (ul)
Clear Color Buffer	12
dNTP's	1.2
Primers: 1 + 2	12 (6 each)
Template DNA	6
Phusion	1.25
ddH₂O	77.5
Total Volume	125

PCR Tube Labeling:

1 = fwd primer + rev primer RBS

2 = fwd primer + rev primer Hsp60 (without RBS)

New PCR Condition:

	Time
98	30 secs
98	10 secs
64	15 secs
72	15 secs
72	5 mins
4	hold

Some housekeeping tasks

Monday, June 30, 2014

0.8% Agarose Gel:

25 mL 1x TBE buffer

0.2 g Agarose

1uL EtBr

100 mL LB and autoclaved:

1g tryptone

0.5g yeast extract

1g NaCl

Loaded 1 kb of volume 1 ul and then 6.25 ul of samples (w/RBS in lane 2 and w/o RBS lane 3)

Gel started at 3:30 pm

Gel stopped at 4:40 pm

Made 10, 5 mL LB+AMP, E. coli dam- pBRES36a culture at 6 pm

Mini-Prep

Tuesday, July 1, 2014

Mini-prepped 50 mL of E. coli dam- pBRES36a in LB+AMP

Obtained:

450 ul of 17.6 ng/ul of DNA

50 ul of 14.6 ng/ul of DNA

Primer Walking and PCR Purification

Wednesday, July 2, 2014

Primer Walking:

Belle submitted the plasmid (pBRES36a) to GeneWiz for Primer Walking

PCR Purification:

Purified 45 mL of both RBS and no RBS PCR products using new SV Wizard Kit from Promega

Added 75 mL of 95% EtOH to membrane wash solution

Final Concentration:

Tube Labeling	PCR Product	Concentration (ng/uL)
1	RBS	17.0
2	No RBS	20.9

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 <br />
 </div>
-<div id = "page_80">
-<p>Restriction Digest of PCR Product with XbaI and PstI</p>
-<p>Thursday, July 3, 2014</p>
-<p>Result</p>
-<p>1. 8.6 ug/mL for RBS PCR product</p>
-<p>2. 11.5 ug/mL for no RBS PCR product</p>
-<br />
-<p>Mini-Prep</p>
-</div>
-<div id = "page_81">
-<p>Tuesday, July 8, 2014</p>
-<p>Mini-prepped 5x5 mL of E. coli dam- pBRES36a in LB+amp</p>
-<p>Ran out of lysis solution ,unable to mini prep the remaining 25 mL</p>
-<p>Yield:</p>
-<p>5x50 mL = 250 mL </p>
-<p>Concentration: 42 ug/mL, equivalent to10.5 ng plasmid</p>
-<br />
-<p>Testing old Tubes and Preparing LB Agar (100mL)</p>
-</div>
-<div id = "page_82">
-<p>Wednesday, July 9, 2014</p>
-<p>Testing Old Mini-prep Columns and Tubes:</p>
-<p>Use 20 uL of 3.0 ug/mL DNA</p>
-<p>Result</p>
-<p>New tubes: 52 ug/mL</p>
-<p>Old Tubes: 20 ug/mL</p>
-<p>Conclusion: old tubes can no longer be used purchased new tubes </p>
-<p>100 mL LB Agar and autoclaved:</p>
-<p>1g tryptone</p>
-<p>0.5g yeast extract</p>
-<p>1g NaCl</p>
-<p>1.5 g Agar (1.5%)</p>
-<p>100 mL Water (ddH2O) </p>
-<p>50 mL aliquots </p>
-<b><br />
-</div>
-<div id = "page_83">
-</b><p><b>Ligating Hsp60 Inserts Into Vector Backbone</b></p>
-<p>Monday, July 14, 2014</p>
-<p>1. Digestion</p>
-<br />
-<p>Materials:</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>DNA</p></td>
-<td valign="top" ><p>40</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>XbaI restriction enzyme</p></td>
-<td valign="top" ><p>1.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>PstI restriction enzyme</p></td>
-<td valign="top" ><p>1.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Total</p></td>
-<td valign="top" ><p>50</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_84">
-<p>Procedures:</p>
-<p>1. Mixed materials together in microcentrifuge tubes</p>
-<p>2. Incubate 30 minutes at 37 degrees Celsius</p>
-<br />
-<p>2. PCR Purification (using Promega Wizard SV gel clean up kit)</p>
-<p>3. Ligations</p>
-<p>a. Ligation 1: RBS and Vector</p>
-<p>b. Ligation 2: no RBS and Vector</p>
-<br />
-<p>Materials for Ligation 1:</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of RBS insert</p></td>
-<td valign="top" ><p>4</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>9.5</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_85">
-<p>Materials for Ligation 2</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of no RBS insert</p></td>
-<td valign="top" ><p>3</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>10.5</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-<p>Procedures:</p>
-<p>1. 15 minutes at room temperature</p>
-<p>2. Put on ice until transformation</p>
-<p>4. Transformation (see transformation protocol)</p>
-<br />
-</div>
-<div id = "page_86">
-<p>Housekeeping</p>
-<p>Monday, July 14, 2014</p>
-<p>30 mL LB Agar and autoclaved:</p>
-<p>0.3 g tryptone</p>
-<p>0.18 g yeast extract</p>
-<p>0.3 g NaCl</p>
-<p>0.45 g Agar (1.5%)</p>
-<p>30 mL Water (ddH2O) </p>
-<p>Chloramphenicol (CAM)</p>
-<p>10 mg in 1 mL of 95% EtOH</p>
-<p>5ug/mL workign concentration is needed</p>
-<p>Used 35 ug/mL final</p>
-<p>Prep 50 mL LB agar +CAM:</p>
-<p>0.5 g tryptone</p>
-<p>0.25 g yeast extract</p>
-<p>0.5 g NaCl</p>
-<p>0.75 g Agar (1.5%)</p>
-<p>48 mL Water (ddH2O) </p>
-<p>25 uL of 10 mg/mL CAM</p>
-<p>Competent cells growing</p>
-<br />
-</div>
-<div id = "page_87">
-<p>Housekeeping and Ligation</p>
-<p>Wednesday, July 16, 2014</p>
-<p>Prep 75 m</p>
-<p>0.75 g tryptone</p>
-<p>0.375 g yeast extract</p>
-<p>0.5 g NaCl</p>
-<p>1.125 g Agar (1.5%)</p>
-<p>50 mL Water (ddH2O) </p>
-<p>Ligations</p>
-<p>Ligation 1: RBS and Vector</p>
-<p>Ligation 2: no RBS and Vector</p>
-<br />
-</div>
-<div id = "page_88">
-<p>Materials for Ligation 1:</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of RBS insert</p></td>
-<td valign="top" ><p>4</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>9.5</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_89">
-<p>Materials for Ligation 2</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of no RBS insert</p></td>
-<td valign="top" ><p>3</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>10.5</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_90">
-<p>Transformation:</p>
-<p>50 mL comp cells + 5 uL plasmid (shipment plasmid + RBS/no RBS)</p>
-<p>On ice for 2 minutes</p>
-<p>Heat shock 42 degree Celsius for 45 seconds</p>
-<p>Ice 2 minutes</p>
-<p>550 mL SOC media into mixture</p>
-<p>Shake at 37 degree Celsius for 1 hour (recovery)</p>
-<p>Plate onto CAM plate + incubate at 4 pm</p>
-<br />
-</div>
-<div id = "page_91">
-<p>Plan + CAM plate</p>
-<p>Friday, July 18, 2014</p>
-<p>Plan of Attack with pBRES36a</p>
-<p>1. Digest with individual restriction enzymes and a negative control</p>
-<p>a. No enzyme</p>
-<p>b. XbaI</p>
-<p>c. PstI</p>
-<p>d. SpeI</p>
-<p>e. EcoRI</p>
-<p>2. Visualize on Gel</p>
-<p>3. Double Digest with Pair from Step 1</p>
-<p>4. Visualize on gel</p>
-<p>5. Map the sequence</p>
-<p><br />
-</p>
-<p>CAM plates made with 35 ug/mL</p>
-<p>Transformation with:</p>
-<p>1. Ligation product (2 of them)</p>
-<p>2. Plasmid that's known to express CAM (for control)</p>
-<br />
-</div>
-<div id = "page_92">
-<p>7/20/14</p>
-<p>2<sup>nd</sup> Double Digestion: For the purpose of confirming that there was no mix up in the previous digestion.</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Components</p></td>
-<td valign="top" ><p>Amount (uL)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>10x Fast Digest Buffer</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>pBRES36a (42ng/uL)</p></td>
-<td valign="top" ><p>16</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>SpeI</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>PstI</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Total</p></td>
-<td valign="top" ><p>20</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_93">
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Lane # (from left)</p></td>
-<td valign="top" ><p>Sample</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>Ladder (not visible)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>No enzyme</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>3</p></td>
-<td valign="top" ><p>PstI and SpeI</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>4</p></td>
-<td valign="top" ><p>No enzyme</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>PstI and SpeI</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_94">
-<p>Housekeeping</p>
-<p>Wednesday, July 23, 2014</p>
-<p>Cam plates: 35 ug/mL, dilute 1:1000</p>
-<p>Used 75 mL, used 75 ul</p>
-<p>For 4 plates</p>
-<p>Transformation with RBS:</p>
-<p>50 mL E. coli comp cells, 5 uL RBS ligation</p>
-<p>50 mL E. coli comp cells, 5 uL no RBS ligation</p>
-<p>Transformed and recovered for 1 hour</p>
-<p>Plated 50 uL of transformed cells onto cam plates</p>
-<b><br />
-</b><p><b>Restriction Digest of pBRES36a Plasmid</b></p>
-</div>
-<div id = "page_95">
-<p>Monday, July 28, 2014</p>
-<p><b>1. Digestion</b></p>
-<p><b>Purpose</b>: cut with restriction enzymes to provide a rough map for the pBRES36a plasmid and confirm the plasmid's identity</p>
-<b><br />
-</b><p><b>Materials:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagents</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>pBRES36a plasmid</p></td>
-<td valign="top" ><p>17</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Restriction Enzyme</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Total Volume</p></td>
-<td valign="top" ><p>20</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_96">
-<p><b>Procedures:</b></p>
-<p>1. Mix reagents together and spin down gently.</p>
-<p>2. Incubated for 10 minutes</p>
-<p>3. Pipet up and down gently to mix samples</p>
-<p>4. Load each sample onto the agarose gel</p>
-<p>5. Run electrophoresis according to the conditions specified. </p>
-<p><b>Enzyme Used:</b></p>
-<p>1. XbaI</p>
-<p>2. PstI</p>
-<p>3. SpeI</p>
-<p>4. EcoRI</p>
-<b><br />
-</b><p><b>2. Gel Electrophoresis</b></p>
-<br />
-</div>
-<div id = "page_97">
-<p>Run 1: Gel Set Up (7/18/14)</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>lane</p></td>
-<td valign="top" ><p>Reaction/Reagent</p></td>
-<td valign="top" ><p>Amount loaded (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>1 kb Marker</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>Plasmid + XbaI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>3</p></td>
-<td valign="top" ><p>Plasmid + PstI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>4</p></td>
-<td valign="top" ><p>Empty Lane</p></td>
-<td valign="top" ><p>0</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>Plasmid + SpeI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>6</p></td>
-<td valign="top" ><p>Plasmid +EcoRI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>7</p></td>
-<td valign="top" ><p>Plasmid only</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_98">
-<p>Run 1 Condition:</p>
-<p>1. 2.0 hours</p>
-<p>2. 80 Volts</p>
-<p>3. 0.8% Agarose Gel</p>
-<p>4. 1x TBE Buffer</p>
-<p>Run 2: Gel Set Up (7/28/14)</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>lane</p></td>
-<td valign="top" ><p>Reaction/Reagent</p></td>
-<td valign="top" ><p>Amount loaded (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>1 kb Marker</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>Plasmid + XbaI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>3</p></td>
-<td valign="top" ><p>Plasmid + PstI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>4</p></td>
-<td valign="top" ><p>Plasmid + SpeI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>Plasmid +EcoRI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>6</p></td>
-<td valign="top" ><p>Plasmid +SpeI and EcoRI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>7</p></td>
-<td valign="top" ><p>Hsp60 + XbaI and PstI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>8</p></td>
-<td valign="top" ><p>mCherry + XbaI and PstI</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-</tbody>
-</table>
-<p>Run 2 Condition:</p>
-<p>1. 1.5 hours</p>
-<p>2. 80 Volts</p>
-<p>3. 0.8% Agarose Gel</p>
-<p>4. 1x TBE Buffer</p>
-<br />
-</div>
-<div id = "page_99">
-<p>July 28, 2014 con’t:</p>
-<p>Results</p>
-<p>Run 1 (7/18/14) Left and Run 2 (7/28/14) Right</p>
-<br />
-<p>Ligation, Transformation, Selection</p>
-<p>Thursday, July 24, 2014</p>
-<p>Ligations</p>
-<p>Ligation 1: RBS and Vector</p>
-<p>Ligation 2: no RBS and Vector</p>
-<br />
-<p>Materials for Ligation 1:</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of RBS insert</p></td>
-<td valign="top" ><p>4</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>9.5</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_100">
-<p>Materials for Ligation 2</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Reagent</p></td>
-<td valign="top" ><p>Amount (ul)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Ligase</p></td>
-<td valign="top" ><p>1</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Buffer T4</p></td>
-<td valign="top" ><p>2</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>30 ng of no RBS insert</p></td>
-<td valign="top" ><p>3</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>60 ng of vector plasmid</p></td>
-<td valign="top" ><p>3.5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH2O (Volume to 20)</p></td>
-<td valign="top" ><p>10.5</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_101">
-<p>Incubate at RT for 15 minutes</p>
-<p>Transformation</p>
-<p>1. 50 mL E. coli comp cells</p>
-<p>2. 6 uL plasmid</p>
-<p>3. 2 min on ice</p>
-<p>4. 45s heat shock at 42 degrees Celsius</p>
-<p>5. 2 min on ice</p>
-<p>6. 950 mL SOC growth media</p>
-<p>7. 1 hour recovery at 37 degrees Celsius</p>
-<p>Selection:</p>
-<p>plate 60 mL of transformed cells onto selection plate (LB + CAM plates)</p>
-<br />
-</div>
-<div id = "page_102">
-<p>8/1/14</p>
-<p>1. Take out overnight liquid culture around 11:00 am</p>
-<p>2. Mini prep iGEM plasmid parts</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Part</p></td>
-<td valign="top" ><p>Amount (ng/uL)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Desaturase</p></td>
-<td valign="top" ><p>42.6</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Cathelicidin</p></td>
-<td valign="top" ><p>12.7</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>mCherry</p></td>
-<td valign="top" ><p>12.7</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_103">
-<p>8/4/14 Lab</p>
-<p><b>Make 1% Gel (30 mL):</b></p>
-<p>0.3 g Agarose</p>
-<p>30 mL 1x TBE</p>
-<p>1 uL EtBr</p>
-<p><b>Restriction Digests:</b></p>
-<p>hsp60 PCR products:</p>
-<p>RBS: no RBS:</p>
-<p>6 uL ddH<sub>2</sub>O 6 uL ddH<sub>2</sub>O</p>
-<p>2 uL Buffer 2 uL Buffer</p>
-<p>1 uL XbaI 1 uL XbaI</p>
-<p>1 uL PstI 1 uL PstI</p>
-<p>10 uL PCR product 10 uL PCR product</p>
-<p>iGEM Parts:</p>
-<p>mCherry: Cathelicidin: deSaturase:</p>
-<p>6 uL ddH<sub>2</sub>O 6 uL ddH<sub>2</sub>O 6 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green Buffer 2 uL FD Green Buffer 2 uL FD Green Buffer</p>
-<p>1 uL XbaI 1 uL XbaI 1 uL XbaI</p>
-<p>1 uL PstI 1 uL PstI 1 uL PstI</p>
-<p>10 uL mCherry 10 uL Cathelicidin 10 uL deSaturase</p>
-<p><b>PCR Purification:</b></p>
-<p>Run PCR purification of hsp60 digests (RBS and no RBS)</p>
-<p>1: RBS concentration-</p>
-<p>2: no RBS concentration –</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Ladder – mcherry – cath – desat - empty - cath – mcherry – desat</p>
-<p>Failed. Will upload pic later</p>
-<p><b>Ligation:</b></p>
-<p>Ligate hsp60 RBS and no RBS into pSB1C3 backbones</p>
-<p>RBS: no RBS:</p>
-<p>9.5 uL ddH<sub>2</sub>O 10.5 uL ddH<sub>2</sub>O</p>
-<p>3.5 uL backbone 3.5 uL backbone</p>
-<p>4 uL insert 3 uL insert</p>
-<p>2 uL buffer 2 uL buffer</p>
-<p>1 uL ligase 1 uL ligase</p>
-</div>
-<div id = "page_104">
-<p><b>Transformation:</b></p>
-<p>Transform RBS and no RBS ligations into <i>E. coli </i>competent cells</p>
-<p>5 uL plasmid</p>
-<p>100 uL competent cells</p>
-<p>Ice 2 minutes</p>
-<p>Heat shock (42C) 45s</p>
-<p>Ice 2 minutes</p>
-<p>Add 950 uL outgrowth media</p>
-<p>Recover with shaking (37C) for 1 hour</p>
-<p>Plate onto selection plate (chloramphenicol)</p>
-<p><b>Restriction Digest:</b></p>
-<p>Cut mCherry prep, Cath. Prep., deSat. Prep, 1 RBS, 1 no RBS, 1 mCherry with XbaI and PstI</p>
-<p>6 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green Buffer</p>
-<p>1 uL XbaI</p>
-<p>1 uL PstI</p>
-<p>10 uL plasmid DNA</p>
-<p><b>Gel Electrophoresis:</b></p>
-</div>
-<div id = "page_105">
-<p>1 kb+ ladder – mCherry prep – Cath. Prep – deSat prep – 1 RBS – 1 no RBS – 1 mCherry - </p>
-<table cellpadding="0" cellspacing="0" align="left">
-<tbody>
-<tr>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td></td>
-</tr>
-</tbody>
-</table>
-<br />
-<br />
-</div>
-<div id = "page_106">
-<p>8/5/15 Lab</p>
-<p><b>Make 1% Gel (35 mL)</b></p>
-<p>0.35 g Agarose</p>
-<p>35 mL 1x TBE</p>
-<p>1 uL EtBR</p>
-<p><b>Restriction Digests:</b></p>
-<p>Master Mix (for 6 reactions): x6.5 =</p>
-<p>6 uL ddH<sub>2</sub>O 39 uL</p>
-<p>2 uL Green Buffer 13 uL</p>
-<p>1 uL XbaI 6.5 uL</p>
-<p>1 uL PstI 6.5 uL</p>
-<p>Combine 10 uL of Master Mix with 10 uL of each plasmid:</p>
-<p>mCherry Prep, Cath. Prep, deSat prep., 1 RBS, 1 no RBS, 1 mCherry</p>
-<p>Incubate 45 mins at 37C</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Run 1 hour 30 mins</p>
-</div>
-<div id = "page_99">
-<p>Lanes:</p>
-<p>1 kb+ ladder – mCherry mini – Desat – Cath – 1 RBS – 1 no RBS – 1 mCherry – 1 kb+ ladder</p>
-<table cellpadding="0" cellspacing="0" align="left">
-<tbody>
-<tr>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td></td>
-</tr>
-</tbody>
-</table>
-<br />
-<br />
-</div>
-<div id = "page_108">
-<p>08/08/14</p>
-<p>Used the culture collected from 8/1/14 mini-prep for the subsequent restriction digest</p>
-<p>1. Restriction digest (45 minutes)</p>
-<p>2. Run agarose gel</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td> </td>
-<td><p>Plasmid</p></td>
-<td><p>Expected Band (kb)</p></td>
-</tr>
-<tr>
-<td><p>1</p></td>
-<td rowspan="2" ><p>Desaturase</p></td>
-<td rowspan="2" ><p>~1</p></td>
-</tr>
-<tr>
-<td><p>2</p></td>
-</tr>
-<tr>
-<td><p>3</p></td>
-<td rowspan="2" ><p>Cathelicidin</p></td>
-<td rowspan="2" ><p>~0.1</p></td>
-</tr>
-<tr>
-<td><p>4</p></td>
-</tr>
-<tr>
-<td><p>5</p></td>
-<td rowspan="2" ><p>RBS</p></td>
-<td rowspan="2" ><p>~0.3</p></td>
-</tr>
-<tr>
-<td><p>6</p></td>
-</tr>
-<tr>
-<td><p>7</p></td>
-<td rowspan="2" ><p>No RBS</p></td>
-<td rowspan="2" ><p>~0.3</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>8</p></td>
-</tr>
-</tbody>
-</table>
-<p>Digestion was incomplete and need to repeat experiment</p>
-<p>Experiment was repeated on 8/9/14 and digestion was successful. The desire bands were excised and purified. Unfortunately the image file was lost due to a technical problem prior to routine file backup. However, further experiments continued to use parts isolated on this date and bands of correct size were present through all of these steps. </p>
-<br />
-</div>
-<div id = "page_109">
-<p><b>Lab Work 8/12/14:</b></p>
-<p><b>Gel Purification:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>Gel Fragment</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>Gel + Tube Mass (g)</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>Tube Mass (g)</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>Gel Mass (g)</p></td>
-<td width="15%" valign="bottom" ><p>Membrane Binding Solution Added (uL)</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>DNA Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>1 Cath. Part</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.133</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.083</p></td>
-<td width="15%" nowrap="" ><p>83</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>2.9</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>2 Cath. Part</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.146</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.096</p></td>
-<td width="15%" nowrap="" ><p>96</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.9</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>3 Desat. Part</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.173</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.123</p></td>
-<td width="15%" nowrap="" ><p>123</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.1</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>4 Desat. Part</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.192</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.142</p></td>
-<td width="15%" nowrap="" ><p>142</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.1</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>1 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.160</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.110</p></td>
-<td width="15%" nowrap="" ><p>110</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.2</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>2 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.152</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.102</p></td>
-<td width="15%" nowrap="" ><p>102</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.2</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>3 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.151</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.101</p></td>
-<td width="15%" nowrap="" ><p>101</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>3.2</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>4 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.200</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.150</p></td>
-<td width="15%" nowrap="" ><p>150</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>4.3</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>5 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.201</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.151</p></td>
-<td width="15%" nowrap="" ><p>151</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>6.6</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p><s>6 Backbone</s></p></td>
-<td width="17%" nowrap="" valign="bottom" ><p><s>1.058</s></p></td>
-<td width="14%" nowrap="" valign="bottom" ><p><s>1.050</s></p></td>
-<td width="13%" nowrap="" valign="bottom" ><p><s>0.008</s></p></td>
-<td width="15%" nowrap="" ><p><s>8</s></p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>N/A</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>7 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.243</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.193</p></td>
-<td width="15%" nowrap="" ><p>193</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>5.7</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>8 Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.159</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.109</p></td>
-<td width="15%" nowrap="" ><p>109</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>5.7</p></td>
-</tr>
-<tr>
-<td width="16%" nowrap="" valign="bottom" ><p>Backbone</p></td>
-<td width="17%" nowrap="" valign="bottom" ><p>1.300</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>1.050</p></td>
-<td width="13%" nowrap="" valign="bottom" ><p>0.250</p></td>
-<td width="15%" nowrap="" ><p>250</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>4.4</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_110">
-<p>*Add 10 uL of Membrane Binding Solution per 10 mg of Gel slice</p>
-<p>-Vortex Gel and solution</p>
-<p>-Incubate at 55C for 10 mins to melt gel</p>
-<p>-Spin Down</p>
-<p>-Gel Purify</p>
-<p><b>Miniprep:</b></p>
-<p>Miniprepped mRFP1 and MelA liquid cultures</p>
-<p>-used same elution buffer for two of the same sample to obtain double DNA concentration</p>
-<p>Concentrations:</p>
-<p>1 mRFP: 516 ng/uL</p>
-<p>2 mRFP: 462.5 ng/uL</p>
-<p>3 mRFP: 243.5 ng/uL</p>
-<p>1 MelA: 117.9 ng/uL</p>
-<p>2 MelA: 158.3 ng/uL</p>
-<p>3 MelA: 146.1 ng/uL</p>
-<br />
-<p><b>Lab Work 8/13/14:</b></p>
-<p><b>Restriction Digest:</b></p>
-<p>Digesting 1 mRFP, 2 mRFP, 3 mRFP, 1 MelA, 2 MelA, 3 MelA with XbaI and PstI.</p>
-<p>20 uL Reactions: Master Mix:</p>
-<p>5 uL Plasmid DNA 13 uL FD Green Buffer</p>
-<p>2 uL FD Green Buffer 6.5 uL XbaI</p>
-<p>1 uL XbaI 6.5 uL PstI</p>
-<p>1 uL PstI 78 uL ddH<sub>2</sub>O</p>
-<p>12 uL ddH<sub>2</sub>O 104 uL Total</p>
-<p>*Reactions are 5 uL Plasmid + 15 uL Master Mix</p>
-<p>*There are 6 reactions. Master Mix is 6.5x</p>
-<p>*Incubate for 45 minutes at 37C</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Running a gel to check the validity of the mRFP1 and MelA mini-prepped DNA.</p>
-<p>Expect to see mRFP1 band at 706 bp and MelA band at 1844 bp.</p>
-<p>Lanes:</p>
-<p>1 kb+ --- 1 mRFP --- 2 mRFP --- 3 mRFP --- 1 MelA --- 2 MelA --- 3 MelA --- EMPTY</p>
-<p>Gel:</p>
-</div>
-<div id = "page_111">
-<p><b>Gel Purification:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td nowrap="" valign="bottom" ><p>Gel Fragment</p></td>
-<td nowrap="" valign="bottom" ><p>Gel + Tube Mass (g)</p></td>
-<td nowrap="" valign="bottom" ><p>Tube Mass (g)</p></td>
-<td nowrap="" valign="bottom" ><p>Gel Mass (g)</p></td>
-<td valign="bottom" ><p>Membrane Binding Solution Added (uL)</p></td>
-<td valign="bottom" ><p>DNA Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>1 mRFP</p></td>
-<td nowrap="" valign="bottom" ><p>1.142</p></td>
-<td nowrap="" valign="bottom" ><p>1.017</p></td>
-<td nowrap="" valign="bottom" ><p>0.125</p></td>
-<td nowrap="" valign="bottom" > </td>
-<td nowrap="" valign="bottom" > </td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>2 mRFP</p></td>
-<td nowrap="" valign="bottom" ><p>1.143</p></td>
-<td nowrap="" valign="bottom" ><p>1.009</p></td>
-<td nowrap="" valign="bottom" ><p>0.134</p></td>
-<td nowrap="" valign="bottom" > </td>
-<td nowrap="" valign="bottom" > </td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>3 mRFP</p></td>
-<td nowrap="" valign="bottom" ><p>1.149</p></td>
-<td nowrap="" valign="bottom" ><p>1.012</p></td>
-<td nowrap="" valign="bottom" ><p>0.137</p></td>
-<td nowrap="" valign="bottom" > </td>
-<td nowrap="" valign="bottom" > </td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_112">
-<p>*Did not complete Gel Purification</p>
-<p><b>Plan for Tomorrow:</b></p>
-<p>1) Finish Gel Purification (Possibly Re-Run. I believe I cut wrong fragment)</p>
-<p>2) Make chloramphenicol plates</p>
-<p>3) Resuspend YF1 &amp; FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)</p>
-<p>4) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)</p>
-<p>5) Transform and Plate (YF1 &amp; FixJ) and FixK2 Promoter</p>
-<p>If able to obtain Kanamycin:</p>
-<p>1) Make Kanamycin plates (2-3)</p>
-<p>2) Transform and Plate mCherry Bomb (3.4 uL of plasmid left)</p>
-<br />
-</div>
-<div id = "page_113">
-<p><b>Lab Work 8/14/14:</b></p>
-<p><b>Plan:</b></p>
-<p>1) Re-Run mRFP1 on Gel and then Gel Purify</p>
-<p>2) Make Chloramphenicol Plates</p>
-<p>3) Dilute mCherry Bomb and nanodrop</p>
-<p><s>3a) If enough DNA is present, run PCR </s>(1.4 ng/uL only)</p>
-<p>3b) If not enough DNA is present, make Kanamycin plates</p>
-<p>4) Resuspend YF1 &amp; FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)</p>
-<p>5) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)</p>
-<p>6) Transform and Plate (YF1 &amp; FixJ) and FixK2 Promoter (Chloramphenicol)</p>
-<p>7) Transform and Plate mCherry Bomb plasmid (Kanamycin)</p>
-<p><b>mRFP1 Gel Electrophoresis:</b></p>
-<p>-Create 25 mL or 1% agarose gel and let solidify</p>
-<p>-Load digested mRFP1 DNA from 8/13/14</p>
-<p>Lanes:</p>
-<p>1 kb+ Ladder --- Empty --- 1 mRFP --- empty --- 2 mRFP --- empty --- 3 mRFP --- empty</p>
-<p><b>Dilute mCherry Bomb and nanodrop:</b></p>
-<p>-1 uL mCherry Bomb into 9 uL ddH<sub>2</sub>O</p>
-<p>-Concentration of 1.4 ng/uL</p>
-<p><b>Make Plates:</b></p>
-<p>*1 Kanamycin; 3 Chloramphenicol</p>
-<p>-Kanamycin working concentration of 50 ng/mL</p>
-<p>-CAM working concentration of 25 ng/mL</p>
-</div>
-<div id = "page_114">
-<p><b>Resuspend DNA:</b></p>
-<p>*Added 10 uL ddH<sub>2</sub>O to each part to resuspend</p>
-<p><b>Transform:</b></p>
-<p>Transformed Blue Light Sensor, Blue Light Promoter, and mCherry Bomb</p>
-<p>*Plates put in 37C incubator at 4:16 PM</p>
-<p><b>Gel Purification:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="15%" nowrap="" ><p>Gel Fragment</p></td>
-<td width="22%" nowrap="" ><p>Gel + Tube Mass (g)</p></td>
-<td width="16%" nowrap="" ><p>Tube Mass (g)</p></td>
-<td width="14%" nowrap="" ><p>Gel Mass (g)</p></td>
-<td width="13%" ><p>Membrane Binding Solution Added (uL)</p></td>
-<td width="17%" ><p>DNA Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td width="15%" nowrap="" ><p>1 mRFP</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>1.195</p></td>
-<td width="16%" nowrap="" ><p>1.014</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>0.181</p></td>
-<td width="13%" nowrap="" ><p>181</p></td>
-<td width="17%" nowrap="" ><p>3.6</p></td>
-</tr>
-<tr>
-<td width="15%" nowrap="" ><p>2 mRFP</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>1.217</p></td>
-<td width="16%" nowrap="" ><p>1.002</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>0.215</p></td>
-<td width="13%" nowrap="" ><p>215</p></td>
-<td width="17%" nowrap="" ><p>2.0</p></td>
-</tr>
-<tr>
-<td width="15%" nowrap="" ><p>3 mRFP</p></td>
-<td width="22%" nowrap="" valign="bottom" ><p>1.158</p></td>
-<td width="16%" nowrap="" ><p>1.012</p></td>
-<td width="14%" nowrap="" valign="bottom" ><p>0.146</p></td>
-<td width="13%" nowrap="" ><p>146</p></td>
-<td width="17%" nowrap="" ><p>4.6</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_115">
-<p><b>Lab Work 8/15/15:</b></p>
-<p><b>Morning:</b></p>
-<p>Took out agar plates of Blue Light Sensor, Blue Light Promoter, and mCherry Bomb</p>
-<p>*Colonies were spotted on each Plate</p>
-<p><b>Evening:</b></p>
-<p>Made liquid cultures for Blue Light Sensor, Blue Light Promoter, and mCherry Bomb</p>
-<p>*5 mL per liquid culture</p>
-<p>*2 Liquid cultures per plasmid</p>
-<p>*Added CAM to Blue Light Sensor and Blue Light Promoter cultures at working concentration of 25 ng/mL</p>
-<p>*Added Kan to mCherry Bomb culture at working concentration of 50 ng/mL</p>
-<br />
-</div>
-<div id = "page_116">
-<p><b>Lab Work 8/16/14:</b></p>
-<p><b>Miniprep:</b></p>
-<p>-Miniprepped liquid cultures of Blue Light Promoter, Blue Light Sensor, and mCherry Bomb</p>
-<p>Concentrations:</p>
-<p>Blue Light Promoter (1:19G): 44.5 ng/uL</p>
-<p>Blue Light Sensor (1:10N): 36.6 ng/uL</p>
-<p>mCherry Bomb: 35 ng/uL</p>
-<br />
-</div>
-<div id = "page_117">
-<p><b>Lab Work 8/18/14:</b></p>
-<p><b>PCR:</b></p>
-<p>-PCRing hsp60 out of plasmid mCherry Bomb</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td nowrap="" colspan="3" valign="bottom" ><p>Master Mix</p></td>
-<td nowrap="" colspan="3" valign="bottom" ><p>Primers and DNA</p></td>
-<td nowrap="" colspan="2" valign="bottom" ><p>PCR Reaction</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>Reagent</p></td>
-<td nowrap="" valign="bottom" ><p>1x Rxn (uL)</p></td>
-<td nowrap="" valign="bottom" ><p>2.5x Rxn (uL)</p></td>
-<td nowrap="" valign="bottom" ><p>Primer</p></td>
-<td nowrap="" valign="bottom" ><p>RBS (uL)</p></td>
-<td nowrap="" valign="bottom" ><p>no RBS (uL)</p></td>
-<td nowrap="" valign="bottom" ><p>Reagent/DNA</p></td>
-<td nowrap="" valign="bottom" ><p>Volume (uL)</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>Buffer</p></td>
-<td nowrap="" valign="bottom" ><p>10</p></td>
-<td nowrap="" valign="bottom" ><p>25</p></td>
-<td nowrap="" valign="bottom" ><p>Fwd Primer</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>Master Mix</p></td>
-<td nowrap="" valign="bottom" ><p>11.5</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>dNTP</p></td>
-<td nowrap="" valign="bottom" ><p>1</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>Rev Primer</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>Primers and DNA</p></td>
-<td nowrap="" valign="bottom" ><p>7.5</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>Phusion</p></td>
-<td nowrap="" valign="bottom" ><p>0.5</p></td>
-<td nowrap="" valign="bottom" ><p>1.25</p></td>
-<td nowrap="" valign="bottom" ><p>mCherry Bomb</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>2.5</p></td>
-<td nowrap="" valign="bottom" ><p>ddH2O</p></td>
-<td nowrap="" valign="bottom" ><p>31</p></td>
-</tr>
-<tr>
-<td nowrap="" valign="bottom" ><p>Total</p></td>
-<td nowrap="" valign="bottom" ><p>11.5</p></td>
-<td nowrap="" valign="bottom" ><p>28.75</p></td>
-<td nowrap="" valign="bottom" ><p>Total</p></td>
-<td nowrap="" valign="bottom" ><p>7.5</p></td>
-<td nowrap="" valign="bottom" ><p>7.5</p></td>
-<td nowrap="" valign="bottom" ><p>Total</p></td>
-<td nowrap="" valign="bottom" ><p>50</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_118">
-<p>*Two 20 uL PCR reactions of each (RBS and no RBS) were carried out</p>
-<p>PCR conditions:</p>
-<p>98C for 30s</p>
-<p><b>98C for 10s</b></p>
-<p><b>66C for 30s x30 cycles</b></p>
-<p><b>72C for 30s</b></p>
-<p>72C for 5 min</p>
-<p>4C holding</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Ran two PCR products with a ladder on a gel. There were no bands shown. PCR was unsuccessful</p>
-<br />
-</div>
-<div id = "page_119">
-<p><b>Lab Work 8/19/14:</b></p>
-<p><b>Restriction Digest:</b></p>
-<p>Digesting Blue Light Sensor and Blue Light Promoter</p>
-<p>20 uL Reactions:</p>
-<p>5 uL Plasmid DNA</p>
-<p>2 uL FD Green Buffer </p>
-<p>1 uL XbaI</p>
-<p>1 uL PstI </p>
-<p>12 uL ddH<sub>2</sub>O </p>
-<p>*Incubate for 45 minutes at 37C</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Running a gel to check the validity of the Blue Light Sensor (1796 bp), Blue Light Promoter (250 bp), and PCR products (~380 bp).</p>
-<p>Lanes:</p>
-<p>1 kb+ ladder --- RBS PCR --- no RBS PCR --- Blue Light Sensor --- Blue Light Sensor --- Blue Light Promoter --- Blue Light Promoter --- empty</p>
-<br />
-</div>
-<div id = "page_120">
-<p>9/12/14</p>
-<p>Goal:</p>
-<p>1. PCR purify Hsp60 with and without RBS</p>
-<p>2. Digestion of purified product with XbaI and PstI</p>
-<p>3. Gel purification of digestion products</p>
-<p>a. Cut out slices and store at 4 ℃</p>
-<p>Experiments:</p>
-<p>Tubes Labeled by MJ</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Tube label</p></td>
-<td><p>Content</p></td>
-</tr>
-<tr>
-<td><p>1, PCR purification product, no RBS</p></td>
-<td><p>1<sup>st</sup> elution: PCR purification product, no RBS</p></td>
-</tr>
-<tr>
-<td><p>2, PCR purification product, no RBS</p></td>
-<td><p>2<sup>nd</sup> elution: PCR purification product, no RBS</p></td>
-</tr>
-<tr>
-<td><p>1, PCR purification product, RBS</p></td>
-<td><p>1<sup>st</sup> elution: PCR purification product, RBS</p></td>
-</tr>
-<tr>
-<td><p>2, PCR purification product, RBS</p></td>
-<td><p>2<sup>nd</sup> elution: PCR purification product, RBS</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_121">
-<p>1. PCR purification</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Step</p></td>
-<td><p>Hsp60 with RBS (uL)</p></td>
-<td><p>Components</p></td>
-<td><p>Hsp60 without RBS (uL)</p></td>
-</tr>
-<tr>
-<td><p>1</p></td>
-<td><p>34.5</p></td>
-<td><p>Membrane binding solution</p></td>
-<td><p>34.5</p></td>
-</tr>
-<tr>
-<td><p>2</p></td>
-<td><p>700</p></td>
-<td><p>Membrane Wash solution</p></td>
-<td><p>700</p></td>
-</tr>
-<tr>
-<td><p>3</p></td>
-<td><p>500</p></td>
-<td><p>Membrane Wash solution</p></td>
-<td><p>500</p></td>
-</tr>
-<tr>
-<td><p>4</p></td>
-<td><p>50</p></td>
-<td><p>1<sup>st</sup> elution with Nuclease free ddH<sub>2</sub>O</p></td>
-<td><p>50</p></td>
-</tr>
-<tr>
-<td><p>5</p></td>
-<td><p>30</p></td>
-<td><p>2<sup>nd</sup> elution with Nuclease free ddH<sub>2</sub>O</p></td>
-<td><p>30</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_122">
-<p>2. Digestion of purified product</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Components</p></td>
-<td><p>Amount for Hsp60 with RBS (uL)</p></td>
-<td><p>Amount for Hsp60 without RBS (uL)</p></td>
-</tr>
-<tr>
-<td><p>10x Fast Digest Green Buffer</p></td>
-<td><p>2</p></td>
-<td><p>2</p></td>
-</tr>
-<tr>
-<td><p>PCR Product of Hsp60</p></td>
-<td><p>7</p></td>
-<td><p>7</p></td>
-</tr>
-<tr>
-<td><p>XbaI</p></td>
-<td><p>1</p></td>
-<td><p>1</p></td>
-</tr>
-<tr>
-<td><p>PstI</p></td>
-<td><p>1</p></td>
-<td><p>1</p></td>
-</tr>
-<tr>
-<td><p>ddH<sub>2</sub>O</p></td>
-<td><p>13</p></td>
-<td><p>13</p></td>
-</tr>
-<tr>
-<td><p>Total</p></td>
-<td><p>24</p></td>
-<td><p>24</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-<p>Incubated at 37 ℃ for 30 minutes</p>
-<br />
-<p>2 reactions each were digested for 1<sup>st</sup> elution of both with and without RBS; 1 reaction each for the 2<sup>nd</sup> elution for both with and without RBS<br />
-</div>
-<div id = "page_123">
-/12/14 continued</p>
-<p>3. Gel Set Up:</p>
-<table border="1" cellspacing="0" cellpadding="0" align="left" >
-<tbody>
-<tr>
-<td valign="top" ><p>Lane</p></td>
-<td valign="top" ><p>Sample</p></td>
-<td valign="top" ><p>Amount (uL)</p></td>
-<td valign="top" ><p>Expected band size (bp)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>1kb DNA Ladder</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>None</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>Elution 1, no RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-<td rowspan="3" valign="top" ><p>~ 300 bp</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>3</p></td>
-<td valign="top" ><p>Elution 1, no RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>4</p></td>
-<td valign="top" ><p>Elution 2, no RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>1kb DNA Ladder</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>None</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>6</p></td>
-<td valign="top" ><p>Elution 1, RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-<td rowspan="3" valign="top" ><p>~300 bp</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>7</p></td>
-<td valign="top" ><p>Elution 1, RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>8</p></td>
-<td valign="top" ><p>Elution 2, RBS digested</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-</tbody>
-</table>
-<p><br />
-</p>
-</div>
-<div id = "page_124">
-<p>4. Mass of tube and tube plus sample</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Tube label</p></td>
-<td><p>Sample (lane #)</p></td>
-<td><p>Mass of tube (g)</p></td>
-<td valign="top" ><p>Mass of Tube plus sample (g)</p></td>
-</tr>
-<tr>
-<td><p>1</p></td>
-<td><p>2</p></td>
-<td><p>1.012</p></td>
-<td><p>1.1</p></td>
-</tr>
-<tr>
-<td><p>2</p></td>
-<td><p>3</p></td>
-<td><p>1.005</p></td>
-<td><p>1.098</p></td>
-</tr>
-<tr>
-<td><p>3</p></td>
-<td><p>4</p></td>
-<td><p>1.018</p></td>
-<td><p>1.121</p></td>
-</tr>
-<tr>
-<td><p>4</p></td>
-<td><p>6</p></td>
-<td><p>1.01</p></td>
-<td valign="top" ><p>1.105</p></td>
-</tr>
-<tr>
-<td><p>5</p></td>
-<td><p>7</p></td>
-<td><p>1.02</p></td>
-<td valign="top" ><p>1.113</p></td>
-</tr>
-<tr>
-<td><p>6</p></td>
-<td><p>8</p></td>
-<td><p>1.003</p></td>
-<td valign="top" ><p>1.094</p></td>
-</tr>
-</tbody>
-</table>
-<p><br />
-</div>
-<div id = "page_125">
-<b>Lab Work 9/17/14:</b></p>
-<p><b>Goals:</b></p>
-<p>1) Digestion of Blue Light Parts w/ X+P</p>
-<p>2) Run Parts on Gel</p>
-<p>3) Gel Purify Blue Light Parts, hsp60 parts, vector backbone</p>
-<p>4) Ligate hsp60 + backbone</p>
-<p><b>Digestion:</b></p>
-<p>9 uL ddH<sub>2</sub>O</p>
-<p>2 uL Fast Digest Green Buffer</p>
-<p>1 uL XbaI</p>
-<p>1 uL PstI</p>
-<p>7 uL Plasmid (Blue Light Sensor and Blue Light Promoter)</p>
-<p>20 uL rxn</p>
-<p>Incubate at 37 C for 30 min</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Lanes:</p>
-<p>1 kb+ ladder – Blue Sensor – Blue Sensor – Blue Promoter – Blue Promoter – empty</p>
-<p>Image at 30 mins: Image at 1 hour, 15 mins:</p>
-<p>*Lanes 4/5 extracted at 30 mins</p>
-<p><b>Purification:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="13%" nowrap="" > </td>
-<td width="15%" nowrap="" ><p>Tube Weight (g)</p></td>
-<td width="20%" nowrap="" ><p>Tube + Gel Weight (g)</p></td>
-<td width="21%" nowrap="" ><p>Gel Fragment Weight (g)</p></td>
-<td width="29%" nowrap="" ><p>Membrane Binding Solution (uL)</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>Backbone</p></td>
-<td width="15%" nowrap="" ><p>1.021</p></td>
-<td width="20%" nowrap="" ><p>1.183</p></td>
-<td width="21%" nowrap="" ><p>0.162</p></td>
-<td width="29%" nowrap="" ><p>162</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>Blue Promoter</p></td>
-<td width="15%" nowrap="" ><p>1.016</p></td>
-<td width="20%" nowrap="" ><p>1.198</p></td>
-<td width="21%" nowrap="" ><p>0.182</p></td>
-<td width="29%" nowrap="" ><p>182</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>Backbone</p></td>
-<td width="15%" nowrap="" ><p>1.016</p></td>
-<td width="20%" nowrap="" ><p>1.187</p></td>
-<td width="21%" nowrap="" ><p>0.171</p></td>
-<td width="29%" nowrap="" ><p>171</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>Blue Sensor</p></td>
-<td width="15%" nowrap="" ><p>1.004</p></td>
-<td width="20%" nowrap="" ><p>1.126</p></td>
-<td width="21%" nowrap="" ><p>0.122</p></td>
-<td width="29%" nowrap="" ><p>122</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>no RBS</p></td>
-<td width="15%" nowrap="" > </td>
-<td width="20%" nowrap="" > </td>
-<td width="21%" nowrap="" ><p>0.088</p></td>
-<td width="29%" nowrap="" ><p>88</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>no RBS</p></td>
-<td width="15%" nowrap="" > </td>
-<td width="20%" nowrap="" > </td>
-<td width="21%" nowrap="" ><p>0.093</p></td>
-<td width="29%" nowrap="" ><p>93</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>RBS</p></td>
-<td width="15%" nowrap="" > </td>
-<td width="20%" nowrap="" > </td>
-<td width="21%" nowrap="" ><p>0.095</p></td>
-<td width="29%" nowrap="" ><p>95</p></td>
-</tr>
-<tr>
-<td width="13%" nowrap="" ><p>RBS</p></td>
-<td width="15%" nowrap="" > </td>
-<td width="20%" nowrap="" > </td>
-<td width="21%" nowrap="" ><p>0.093</p></td>
-<td width="29%" nowrap="" ><p>93</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_125">
-<p><b>Ligation:</b></p>
-<p>RBS no RBS</p>
-<p>1 uL ligase 1 uL ligase</p>
-<p>2 uL Buffer 2 uL Buffer</p>
-<p>4 uL Insert 3 uL Insert</p>
-<p>3.5 uL Backbone 3.5 uL Backbone</p>
-<p>9.5 uL ddH<sub>2</sub>O 10.5 uL ddH<sub>2</sub>O</p>
-<p>20 uL 20 uL</p>
-<br />
-</div>
-<div id = "page_126">
-<p>9/22/14</p>
-<p><b>Goals</b>:</p>
-<p>1. Digestion of desaturase, and mRFP-1</p>
-<p>2. Purification of desaturase and mRFP-1</p>
-<p>3. Ligation into the vector</p>
-<p>4. Digestion of 7 uL of plasmid DNA</p>
-<p>5. Purification</p>
-<p><b>Experiment:</b></p>
-<p>Cut mRPF-1 with EcoRI and XbaI</p>
-<p>Desaturase with EcoRI and SpeI</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Components mRFP-1</p></td>
-<td valign="top" ><p>Amount (uL)</p></td>
-<td valign="top" ><p>Components desaturase</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>10x Fast Digest Buffer</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>10x Fast Digest Buffer</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>plasmid</p></td>
-<td valign="top" ><p>7</p></td>
-<td valign="top" ><p>plasmid</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>EcoRI</p></td>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>EcoRI</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>XbaI</p></td>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>SpeI</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>ddH<sub>2</sub>O</p></td>
-<td valign="top" ><p>9</p></td>
-<td valign="top" ><p>ddH<sub>2</sub>O</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Total</p></td>
-<td valign="top" ><p>19</p></td>
-<td valign="top" ><p>Total</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_127">
-<p><b>Gel</b>:</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td valign="top" ><p>Lane</p></td>
-<td valign="top" ><p>1</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>3</p></td>
-<td valign="top" ><p>4</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Components</p></td>
-<td valign="top" ><p>1kb DNA Ladder</p></td>
-<td colspan="3" valign="top" ><p>Desaturase (EcoRI+SpeI)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Amount (uL)</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Lane</p></td>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>6</p></td>
-<td valign="top" ><p>7</p></td>
-<td valign="top" ><p>8</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Components</p></td>
-<td valign="top" ><p>1kb DNA Ladder</p></td>
-<td colspan="3" valign="top" ><p>mRFP-1 (EcoRI+XbaI)</p></td>
-</tr>
-<tr>
-<td valign="top" ><p>Amount (uL)</p></td>
-<td valign="top" ><p>2</p></td>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>5</p></td>
-<td valign="top" ><p>5</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_128">
-<p>9/24/14</p>
-<p>mRFP-1 and desaturase plasmids were previously collected and this is the resulting digestion (9/22/14) followed by gel purification of the previous experiments.</p>
-<p>1. Gel purification: and resulting concentrations: </p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Tube label</p></td>
-<td><p>Part excised</p></td>
-<td><p>Mass (g)</p></td>
-<td valign="top" ><p>Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td><p>1</p></td>
-<td rowspan="3" ><p>Desaturase Vector Backbone</p></td>
-<td colspan="2" rowspan="3" ><p>Not used since this is the backbone</p></td>
-</tr>
-<tr>
-<td><p>2</p></td>
-</tr>
-<tr>
-<td><p>3</p></td>
-</tr>
-<tr>
-<td><p>4</p></td>
-<td rowspan="3" ><p>Desaturase Part</p></td>
-<td><p>0.124</p></td>
-<td valign="top" ><p>3.9</p></td>
-</tr>
-<tr>
-<td><p>5</p></td>
-<td><p>0.123</p></td>
-<td valign="top" ><p>4.7</p></td>
-</tr>
-<tr>
-<td><p>6</p></td>
-<td><p>0.095</p></td>
-<td valign="top" ><p>4.2</p></td>
-</tr>
-<tr>
-<td><p>7</p></td>
-<td rowspan="3" ><p>mRFP-1 linearized plasmid</p></td>
-<td><p>0.095</p></td>
-<td valign="top" ><p>4.4</p></td>
-</tr>
-<tr>
-<td><p>8</p></td>
-<td><p>0.103</p></td>
-<td valign="top" ><p>4.4</p></td>
-</tr>
-<tr>
-<td><p>9</p></td>
-<td valign="bottom" ><p>0.133</p></td>
-<td valign="top" ><p>4.8</p></td>
-</tr>
-</tbody>
-</table>
-<br />
-</div>
-<div id = "page_129">
-<p>9/26/14</p>
-<p>1. Ligation reaction BH</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td><p>Components</p></td>
-<td><p>Amount (uL)</p></td>
-</tr>
-<tr>
-<td><p>Insert (desaturase)</p></td>
-<td><p>9</p></td>
-</tr>
-<tr>
-<td><p>Vector Backbone (single cut mRFP-1)</p></td>
-<td><p>3.5</p></td>
-</tr>
-<tr>
-<td><p>Ligase</p></td>
-<td><p>1</p></td>
-</tr>
-<tr>
-<td><p>Buffer</p></td>
-<td><p>1.5</p></td>
-</tr>
-<tr>
-<td><p>ddH<sub>2</sub>O</p></td>
-<td><p>5</p></td>
-</tr>
-<tr>
-<td><p>Total</p></td>
-<td><p>20</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_129">
-<p>Negative control: just VB (labeled A)</p>
-<p>Ligation 1 (labeled B)</p>
-<p>Ligation 2 (labeled C)</p>
-<p>2. Transformation into E. coli competent cells BH</p>
-<p>Incubation at 37 ℃ on LB + CAM from 2 pm</p>
-<p>Note: the plate was examined once at 6 hours and another time at 8 hours— yielded no usable colonies </p>
-<p>3. Mini-Prep (BH) of colonies culture grown by MJ</p>
-<p>Labeling maintained the same (1-6)</p>
-<p>Resuspended in 50 uL of elution buffer</p>
-<p>Undetermined concentration</p>
-<p>Ligation and Transformation were repeated 2 times yet the reaction never yielded colonies that survived in LB+CAM culture.</p>
-<br />
-<p><b>Lab Work 9/29/14:</b></p>
-<p><b>Diagnostic Digest:</b></p>
-<p>Hsp60(no RBS) – mRFP1 (~1090 bp) 1) X+P</p>
-<p>Blue Promoter – mRFP1 (~1050 bp) 2) X+P, 3) S+P</p>
-<p>Hsp60 (no RBS) – Blue Sensor (~2160 bp) 4) X+P</p>
-<p>MelA (~1896 bp) 5) X+P</p>
-<p>20 uL Rxns</p>
-<p>9 uL ddH<sub>2</sub>0</p>
-<p>2 uL FD Green</p>
-<p>1 uL XbaI/SpeI</p>
-<p>1 uL PstI</p>
-<p>7 uL Plasmid</p>
-<p>*Incubate for 30 min @ 37C</p>
-</div>
-<div id = "page_130">
-<p><b>Gel Electrophoresis:</b></p>
-<p>1 kb+ ladder – 1 – 2 – 3 – 5 – 4 – empty</p>
-<p>Gel @ 30 mins Gel @ 1 hr, 30 mins</p>
-<table cellpadding="0" cellspacing="0" align="left">
-<tbody>
-<tr>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td align="left" valign="top"></td>
-<td></td>
-<td rowspan="2" align="left" valign="top"></td>
-</tr>
-<tr>
-<td></td>
-</tr>
-</tbody>
-</table>
-<br />
-<p>*Note: Band 3 was extracted after 30 minutes. The lower of band 5 was extracted at 1 hr, 30 min</p>
-<p><b>Gel Extraction:</b></p>
-<p>Blue Promoter – mRFP1 S+P gel weight: 0.086 g</p>
-<p>Hsp60 (no RBS) – Blue Sensor X+P gel weight: 0.062 g</p>
-<p><b>Made 6 CAM plates &amp; 2 AMP plates</b></p>
-<p><b>Transformation:</b></p>
-<p>Transformed RBS (4:1N) and Terminator (4:16G). </p>
-<p>Plate onto AMP plates</p>
-<br />
-</div>
-<div id = "page_131">
-<p><b>Lab Work 10/1/14:</b></p>
-<p><b>Mini Prep:</b></p>
-<p>Mini Prep RBS and Terminator Liquid Cultures</p>
-<p><b>Gel Purify:</b></p>
-<p>Add 86 uL Membrane Binding Solution to Blue Promoter – mRFP1 (S+P) part</p>
-<p>Add 62 uL Membrane Binding Solution to hsp60 (no RBS) – Blue Sensor (X+P) part</p>
-<p><b>Restriction Digests:</b></p>
-<p>Terminator (E+X) (~2150 bp) Cathelicidin (E+S) (~123 bp)</p>
-<p>RBS (E+X) (~2082 bp) Blue Promoter (E+S) (~250 bp)</p>
-<p>20 uL rxns:</p>
-<p>9 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green</p>
-<p>1 uL EcoRI</p>
-<p>1 uL XbaI/SpeI</p>
-<p>7 uL Plasmid</p>
-<p>Incubate 30 mins @ 37C</p>
-</div>
-<div id = "page_132">
-<p><b>Gel Electrophoresis:</b></p>
-<p>Lanes:</p>
-<p>1 kb+ ladder – Terminator (E+X) – Cathelicidin (E+S) – RBS (E+X) – B. Promoter (E+S) – empty</p>
-<table cellpadding="0" cellspacing="0" align="left">
-<tbody>
-<tr>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td></td>
-</tr>
-</tbody>
-</table>
-<br />
-<p>*Run Gel 15 minutes</p>
-<p><b>Gel Purifiy:</b></p>
-<p>RBS (E+X)</p>
-<p>Blue Promoter (E+S)</p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="21%" ><p>Gel Fragment</p></td>
-<td width="13%" ><p>Gel + Tube Mass (g)</p></td>
-<td width="10%" ><p>Tube mass (g)</p></td>
-<td width="10%" ><p>Gel Mass (g)</p></td>
-<td width="21%" ><p>Membrane Binding Solution (uL)</p></td>
-<td width="21%" ><p>DNA Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" ><p><s>Terminator (E+X)</s></p></td>
-<td width="13%" nowrap="" ><p><s> </s></p></td>
-<td width="10%" nowrap="" ><p><s>1.106</s></p></td>
-<td width="10%" nowrap="" ><p><s> </s></p></td>
-<td width="21%" nowrap="" ><p><s> </s></p></td>
-<td width="21%" nowrap="" ><p><s> </s></p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" ><p><s>Cathelicidin (E+S)</s></p></td>
-<td width="13%" nowrap="" ><p><s> </s></p></td>
-<td width="10%" nowrap="" ><p><s>1.015</s></p></td>
-<td width="10%" nowrap="" ><p><s> </s></p></td>
-<td width="21%" nowrap="" ><p><s> </s></p></td>
-<td width="21%" nowrap="" ><p><s> </s></p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" ><p>RBS (E+X)</p></td>
-<td width="13%" nowrap="" ><p>1.033</p></td>
-<td width="10%" nowrap="" ><p>0.985</p></td>
-<td width="10%" nowrap="" ><p>0.048</p></td>
-<td width="21%" nowrap="" ><p>48</p></td>
-<td width="21%" nowrap="" ><p>1.7</p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" ><p>Blue Promoter (E+S)</p></td>
-<td width="13%" nowrap="" ><p>1.082</p></td>
-<td width="10%" nowrap="" ><p>1.005</p></td>
-<td width="10%" nowrap="" ><p>0.077</p></td>
-<td width="21%" nowrap="" ><p>77</p></td>
-<td width="21%" nowrap="" ><p>1.8</p></td>
-</tr>
-</tbody>
-</table>
-<p><b>Ligation:</b></p>
-<p>Blue Promoter – RBS </p>
-<p>15 uL Rxn:</p>
-<p>3.5 uL Insert (Blue Promoter)</p>
-<p>9 uL VB (RBS)</p>
-<p>1.5 uL Buffer</p>
-<p>1 uL Ligase</p>
-<p>0 uL ddH<sub>2</sub>O</p>
-<p><b>Transformation:</b></p>
-<p>Transform Blue Promoter – RBS onto AMP plate</p>
-<br />
-</div>
-<div id = "page_133">
-<p><b>Lab Work 10/4/14:</b></p>
-<p>Morning:</p>
-<p>Made Liquid Cultures of Blue Promoter – RBS part in Turbo E.coli Comp Cells</p>
-<p>Afternoon:</p>
-<p><b>Mini Prep:</b></p>
-<p>Mini Prepped Blue Promoter – RBS : ??? ng/uL</p>
-<p><b>Restriction Digest:</b></p>
-<p>Blue Promoter – RBS (X/P)</p>
-<p>Blue Promoter – RBS (S/P)</p>
-<p>Terminator (S/P)</p>
-<p>20 uL rxns:</p>
-<p>9 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green</p>
-<p>1 uL XbaI/SpeI</p>
-<p>1 uL PstI</p>
-<p>7 uL Plasmid</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Lanes:</p>
-<p>1) 1 kb+ ladder</p>
-<p>2) Blue Promoter – RBS (X+P) (~262 bp)</p>
-<p>3) Blue Promoter – RBS (S+P) (~2350 bp)</p>
-<p>4) Terminator (S+P) (~2150 bp)</p>
-<p><b>Gel Purify:</b></p>
-</div>
-<div id = "page_134">
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="21%" nowrap="" valign="bottom" ><p>Gel Fragment</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>Tube (g)</p></td>
-<td width="12%" nowrap="" valign="bottom" ><p>Gel + Tube (g)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>Gel (g)</p></td>
-<td width="28%" nowrap="" valign="bottom" ><p>Membrane Binding Solution (uL)</p></td>
-<td width="19%" nowrap="" valign="bottom" ><p>Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" valign="bottom" ><p>Blue Promoter-RBS (S/P)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>1.002</p></td>
-<td width="12%" nowrap="" valign="bottom" ><p>1.067</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>0.065</p></td>
-<td width="28%" nowrap="" valign="bottom" ><p>65</p></td>
-<td width="19%" nowrap="" valign="bottom" ><p>5.0</p></td>
-</tr>
-<tr>
-<td width="21%" nowrap="" valign="bottom" ><p>Terminator (S/P)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>1.020</p></td>
-<td width="12%" nowrap="" valign="bottom" ><p>1.093</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>0.073</p></td>
-<td width="28%" nowrap="" valign="bottom" ><p>73</p></td>
-<td width="19%" nowrap="" valign="bottom" ><p>2.1</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_135">
-<p><b>Ligation:</b></p>
-<p>1) Blue Promoter – mRFP1 (S/P) (~3000 bp) <b>+</b> Cathelicidin (X/P) (~120 bp)</p>
-<p>2) Blue Promoter – RBS (S/P) (~2350 bp) <b>+</b> Cathelicidin (X/P) (~120 bp)</p>
-<p>3) Terminator (S/P) (~2150 bp) <b>+</b> hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)</p>
-<p>4) Blue Promoter – mRFP1 (S/P) (~3000 bp) <b>+</b> hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)</p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="10%" nowrap="" ><p>Ligation</p></td>
-<td width="7%" nowrap="" valign="bottom" ><p>VB bp</p></td>
-<td width="23%" nowrap="" valign="bottom" ><p>VB concentration (ng/uL)</p></td>
-<td width="10%" nowrap="" valign="bottom" ><p>Insert bp</p></td>
-<td width="27%" nowrap="" valign="bottom" ><p>Insert concentration (ng/uL)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>VB (uL)</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>Insert (uL)</p></td>
-</tr>
-<tr>
-<td width="10%" ><p>1</p></td>
-<td width="7%" nowrap="" valign="bottom" ><p>3000</p></td>
-<td width="23%" nowrap="" valign="bottom" ><p>2.9</p></td>
-<td width="10%" nowrap="" valign="bottom" ><p>120</p></td>
-<td width="27%" nowrap="" valign="bottom" ><p>3.9</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>4.00</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>8.50</p></td>
-</tr>
-<tr>
-<td width="10%" ><p>2</p></td>
-<td width="7%" nowrap="" valign="bottom" ><p>2350</p></td>
-<td width="23%" nowrap="" valign="bottom" ><p>5.0</p></td>
-<td width="10%" nowrap="" valign="bottom" ><p>120</p></td>
-<td width="27%" nowrap="" valign="bottom" ><p>3.9</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>3.50</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>9.00</p></td>
-</tr>
-<tr>
-<td width="10%" ><p>3</p></td>
-<td width="7%" nowrap="" valign="bottom" ><p>2150</p></td>
-<td width="23%" nowrap="" valign="bottom" ><p>2.1</p></td>
-<td width="10%" nowrap="" valign="bottom" ><p>2160</p></td>
-<td width="27%" nowrap="" valign="bottom" ><p>4.9</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>3.00</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>9.50</p></td>
-</tr>
-<tr>
-<td width="10%" ><p>4</p></td>
-<td width="7%" nowrap="" valign="bottom" ><p>3000</p></td>
-<td width="23%" nowrap="" valign="bottom" ><p>2.9</p></td>
-<td width="10%" nowrap="" valign="bottom" ><p>2160</p></td>
-<td width="27%" nowrap="" valign="bottom" ><p>4.9</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>3.50</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>9.00</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_136">
-<p>1.5 uL Buffer</p>
-<p>1 uL Ligase</p>
-<p><b>Transformation:</b></p>
-<p>Blue Promoter – mRFP1 – Cathelicidin (CAM)</p>
-<p>Blue Promoter – RBS – Cathelicidin (AMP)</p>
-<p>Terminator – hsp60 (no RBS) – Blue Sensor (AMP)</p>
-<p>Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (CAM)</p>
-<br />
-<p><b>Lab Work 10/5/14:</b></p>
-<p><b>Ligation:</b></p>
-<p>Ligation 3: Terminator (S/P) <b>+</b> hsp60 (no RBS) – Blue Sensor (X/P)</p>
-<p>1.5 uL Buffer</p>
-<p>1 uL Ligase</p>
-<p>6 uL VB (Terminator)</p>
-<p>6.5 uL Insert (hsp60-Blue Sensor)</p>
-<p><b>Transformation:</b></p>
-<p>Transform 3: Terminator – hsp60 (no RBS) – Blue Sensor </p>
-<p>AMP Plate</p>
-<p><b>MiniPrep:</b></p>
-<p>Mini 1: Blue Promoter – mRFP1 – Cathelicidin 61.5 ng/uL</p>
-<p>Mini 2: Blue Promoter – RBS – Cathelicidin 85.9 ng/uL</p>
-<p>Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor 81.0 ng/uL</p>
-<p><b>Restriction Digest:</b></p>
-<p>1) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P)</p>
-<p>2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P)</p>
-<p>3) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P)</p>
-<p>4) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P)</p>
-<p>5) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)</p>
-<p>20 uL rxns:</p>
-<p>9 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green</p>
-<p>1 uL XbaI/SpeI</p>
-<p>1 uL PstI</p>
-<p>7 uL Plasmid</p>
-<b><br />
-</div>
-<div id = "page_137">
-</b><p><b>Gel Electrophoresis:</b></p>
-<p>Lanes:</p>
-<p>1) 1 kb+ ladder</p>
-<p>2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P) (~1000 bp) (Part)</p>
-<p>3) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P) (~3100 bp) (VB)</p>
-<p>4) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P) (~370 bp) (Part)</p>
-<p>5) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P) (~2470 bp) (VB)</p>
-<p>6) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)(~3160 bp) (Part)</p>
-<br />
-<p>*After 30 mins, hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp) added to lane 7. Ladder added lane 8.</p>
-</div>
-<div id = "page_138">
-<p><b>Gel Purify:</b></p>
-<table border="1" cellspacing="0" cellpadding="0" width="100%" >
-<tbody>
-<tr>
-<td width="26%" nowrap="" valign="bottom" ><p>Gel Fragment</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>Tube (g)</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>Gel + Tube (g)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>Gel (g)</p></td>
-<td width="26%" nowrap="" valign="bottom" ><p>Membrane Binding Solution (uL)</p></td>
-<td width="18%" nowrap="" valign="bottom" ><p>Concentration (ng/uL)</p></td>
-</tr>
-<tr>
-<td width="26%" nowrap="" valign="bottom" ><p>Mini 1: B.P. - mRFP1 - Cath (S/P)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>1.013</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>1.072</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>0.059</p></td>
-<td width="26%" nowrap="" valign="bottom" ><p>59</p></td>
-<td width="18%" nowrap="" valign="bottom" ><p>2.6</p></td>
-</tr>
-<tr>
-<td width="26%" nowrap="" valign="bottom" ><p>no RBS - Blue Sensor (X/P)</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>1.016</p></td>
-<td width="11%" nowrap="" valign="bottom" ><p>1.065</p></td>
-<td width="8%" nowrap="" valign="bottom" ><p>0.049</p></td>
-<td width="26%" nowrap="" valign="bottom" ><p>49</p></td>
-<td width="18%" nowrap="" valign="bottom" ><p>1.2</p></td>
-</tr>
-</tbody>
-</table>
-</div>
-<div id = "page_139">
-<p><b>Ligation:</b></p>
-<p>1.5 uL Ligation Buffer</p>
-<p>1 uL Ligase</p>
-<p>Lig 1: Lig 2:</p>
-<p>9 uL Blue Promoter (Insert) (~200 bp) 6 uL Terminator (VB) (~2100 bp)</p>
-<p>3.5 uL RBS (VB) (~2100 bp) 6.5 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160)</p>
-<p>Lig 3:</p>
-<p>3.5 uL B. Prom – mRFP1 (VB) (~3100)</p>
-<p>9 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160 bp)</p>
-<p><b>Transformation:</b></p>
-<p>Transform 1: Blue Promoter – RBS onto AMP plate</p>
-<p>Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor onto AMP plate</p>
-<p>Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor onto CAM plate </p>
-<p><b>Make Liquid Cultures:</b></p>
-<p>Made 2 5mL Liquid cultures of LB + AMP, Blue Promoter – RBS </p>
-<br />
-</div>
-<div id = "page_140">
-<p><b>Lab Work 10/6/14:</b></p>
-<p><b>Make Liquid Cultures:</b></p>
-<p>(AMP) Transform 1: Blue Promoter – RBS </p>
-<p>(AMP) Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor </p>
-<p>(CAM) Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor </p>
-<p><b>Mini prep:</b></p>
-<p>Mini 1: Blue Promoter – RBS </p>
-<p>Mini 2: Terminator – hsp60 (no RBS) – Blue Sensor</p>
-<p>Mini 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor</p>
-<p><b>Restriction Digest:</b></p>
-<p>1) Blue Promoter – RBS (X/P)</p>
-<p>2) Blue Promoter – RBS (S/P)</p>
-<p>3) Terminator – hsp60 (no RBS) – Blue Sensor (X/P)</p>
-<p>4) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)</p>
-<p>20 uL Rxns</p>
-<p>9 uL ddH<sub>2</sub>O</p>
-<p>2 uL FD Green</p>
-<p>1 uL XbaI/SpeI</p>
-<p>1 uL PstI</p>
-<p>7 uL Plasmid</p>
-<p><b>Gel Electrophoresis:</b></p>
-<p>Lanes:</p>
-<p>1) 1 kb+ ladder</p>
-<p>2) Blue Promoter – RBS (X/P) (~262 bp)</p>
-<p>3) Blue Promoter – RBS (S/P) (~2341)</p>
-<p>4) Terminator – hsp60 (no RBS) – Blue Sensor (X/P) (~2236)</p>
-<p>5) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P) (~3100)</p>
-<p>*Note: In lane four, the VB is ~2100, will need long separation to see. Part will be bigger than backbone</p>
-<p>*Stop at 15 mins and check lane 2, if correct band is present, extract lane 3 before continuing.</p>
-<p>*Run for another hour and fifteen before stopping a second time and extracting lane 4</p>
-</div>
-<div id = "page_141">
-<table cellpadding="0" cellspacing="0" align="left">
-<tbody>
-<tr>
-<td></td>
-<td></td>
-<td></td>
-<td></td>
-</tr>
-<tr>
-<td></td>
-<td align="left" valign="top"></td>
-<td></td>
-<td align="left" valign="top"></td>
-</tr>
-</tbody>
-</table>
-<br />
-<p><b>Gel Purify:</b></p>
-<p><b>Ligation:</b></p>
-<p><s>Ligation 1: Blue Promoter – RBS (X/P) <b>+</b> Cathelicidin (S/P)</s></p>
-<p>Ligation 2: Blue Promoter – mRFP1 – Cathelicidin (S/P) <b>+</b> Terminator – hsp60 (no RBS) – Blue Sensor (X/P)</p>
-<table border="1" cellspacing="0" cellpadding="0" >
-<tbody>
-<tr>
-<td nowrap="" ><p>Ligation</p></td>
-<td nowrap="" ><p>VB bp</p></td>
-<td><p>VB Concentration (ng/uL)</p></td>
-<td nowrap="" ><p>Insert bp</p></td>
-<td><p>Insert Concentration (ng/uL)</p></td>
-<td nowrap="" ><p>VB (uL)</p></td>
-<td nowrap="" ><p>Insert (uL)</p></td>
-</tr>
-<tr>
-<td nowrap="" ><p><s>1</s></p></td>
-<td nowrap="" ><p><s>2341</s></p></td>
-<td nowrap="" ><p><s> </s></p></td>
-<td nowrap="" ><p><s>123</s></p></td>
-<td nowrap="" ><p><s> </s></p></td>
-<td nowrap="" ><p><s> </s></p></td>
-<td nowrap="" ><p><s> </s></p></td>
-</tr>
-<tr>
-<td nowrap="" ><p>2</p></td>
-<td nowrap="" ><p>2500</p></td>
-<td nowrap="" > </td>
-<td nowrap="" ><p>2236</p></td>
-<td nowrap="" > </td>
-<td nowrap="" > </td>
-<td nowrap="" > </td>
-</tr>
-</tbody>
-</table>
-</div>
 </div>
 <br>