Team:UIUC Illinois/Notebook

From 2014.igem.org

(Difference between revisions)
Line 522: Line 522:
cdh PCR redo:
cdh PCR redo:
-
Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O
+
<ul style="text-indent:30px;">
 +
<li>Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O</li>
 +
</ul><br><br>
Master Mix 1: Regular
Master Mix 1: Regular
<ul style="text-indent:30px;">
<ul style="text-indent:30px;">
-
20uL Buffer G
+
<li>20uL Buffer G</li>
-
4uL forward primer
+
<li>4uL forward primer</li>
-
4uL reverse primer
+
<li>4uL reverse primer</li>
-
0.4uL Q5 polymerase
+
<li>0.4uL Q5 polymerase</li>
-
2uL template
+
<li>2uL template</li>
-
9.6uL H2O
+
<li>9.6uL H2O</li>
 +
</ul><br><br>
 +
Master Mix 2: Forward primer
 +
<ul style="text-indent:30px;">
 +
<li>20uL Buffer G</li>
 +
<li>8uL forward primer</li>
 +
<li>0uL reverse primer</li>
 +
<li>0.4uL Q% polymerase</li>
 +
<li>2uL template</li>
 +
<li>9.6uL H2O</li>
 +
</ul><br><br>
 +
Master Mix 3: Reverse primer
 +
<ul style="text-indent:30px;">
 +
<li>20uL Buffer G</li>
 +
<li>0uL forward primer</li>
 +
<li>8uL reverse primer</li>
 +
<li>0.4uL Q5</li>
 +
<li>2uL template</li>
 +
<li>9.6uL H2O</li>
 +
</ul><br><br>
 +
Master Mix 4: gDNA (10x)
 +
<ul style="text-indent:30px;">
 +
<li>20uL Buffer G</li>
 +
<li>4uL forward primer</li>
 +
<li>4uL reverse primer</li>
 +
<li>0.4uL Q5 polymerase</li>
 +
<li>2uL template</li>
 +
<li>9.6uL H2O</li>
 +
</ul><br><br>
 +
PCR Procedure: Made 4 10uL samples of each master mix
 +
Set up thermocycler with the following settings:
 +
<ul style="text-indent:30px;">
 +
<li>98C: 30 min</li>
 +
<li>40x cycles that ended up being:</li>
 +
<li>98C for 20 minutes</li>
 +
<li>Gradient temperature for 20 minutes: 44.9C, 50.8C. 58.7C, 65.1C</li>
 +
<li>72C for 80 min</li>
 +
<li>72C for 5 minutes</li>
 +
<li>Paused at 4C</li>
 +
</ul><br><br>
 +
Optimized shuttle vector growth environments: From 1 mg/mL concentrate
 +
<ul style="text-indent:30px;">
 +
<li>1mL of each concentration into 2 falcon tubes</li>
 +
<li>Inoculated 3mL LB with cells + Erm stocks</li>
 +
<li>Also had 0ug/mL as a negative control</li>
 +
<li>8 tubes in total</li>
 +
</ul><br><br>
 +
<table width="100%" border="1px solid black" >
 +
<tr>
 +
<th>Concentrations(ug/mL)</th>
 +
<th>H2O(mL)</th>
 +
<th>Erm(mL)</th>
 +
</tr>
 +
<tr>
 +
<td>50</td>
 +
<td>3.8</td>
 +
<td>0.2</td>
 +
</tr>
 +
<tr>
 +
<td>25</td>
 +
<td>1.5</td>
 +
<td>1.5</td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>0.5</td>
 +
<td>0.5</td>
 +
</tr>
 +
</table>
 +
Inoculating Optogene Liquid Cultures:
 +
<ul style="text-indent:30px;">
 +
<li>Added 10mL liquid LB and 10uL chloramphenicol to each of the four falcon tubes</li>
 +
<li>Picked colonies off each labeled plate and added to tubes as follows:
 +
<ul style="text-indent:30px;">
 +
<li>Plate
 +
<ul style="text-indent:30px;">
 +
<li>Green light 1</li>
 +
<li>Green light 2</li>
 +
<li>Red light sensor 1</li>
 +
<li>Red light sensor 2</li>
 +
</ul>
 +
</ul>
 +
<li>Incubated all 4 tubes in 37C shaker at 5:15pm</li>
 +
</ul><br><br>
 +
Inoculated E. coli guaB pdCAF (I4) in 3uL LB + 3uL CM<br><br>
 +
Inoculated Top 10 pdCAF (I5) in 3uL LB + 3uL CM<br><br>
 +
Placed in 37C shaker at 5:15pm<br><br>
 +
</div>
 +
</html>
 +
<html><div id="Tuesday_22nd_July.html"></div></html>
 +
<html>
 +
<div class ="tbnote">
 +
<h2>Tuesday 22<sup>th</sup> July</h2>
 +
<a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a>
 +
<div style="clear: both;"></div>
 +
 +
Made 80uL M9 + glucose solution
 +
<ul style="text-indent:30px;">
 +
Previous solution was contaminated
 +
16uL 5x M9 salts
 +
63.2uL H2O
 +
.4g NH4Cl, .2g NaCl, 1.2g KH2PO4, 2.712g Na2HPO4
 +
16uL MgSO4 + uL CaCl2
 +
.8mL 40% glucose solution using syringe
 +
Stir until dissolved
 +
</ul><br><br>
 +
Erm concentration gradient
 +
<ul style="text-indent:30px;">
 +
Inoculated shuttle vector and top 10 cells
 +
Incubated in 37C
 +
</ul><br><br>
 +
<table width="100%" border="1px solid black" >
 +
<tr>
 +
<th></th>
 +
<th>Erm</th>
 +
<th>LB</th>
 +
</tr>
 +
<tr>
 +
<td>100ug/mL</td>
 +
<td>0.5mL</td>
 +
<td>4.5mL</td>
 +
</tr>
 +
<tr>
 +
<td>50ug/mL</td>
 +
<td>0.25mL</td>
 +
<td>4.75mL</td>
 +
</tr>
 +
<tr>
 +
<td>25ug/mL</td>
 +
<td>0.125mL</td>
 +
<td>4.875mL</td>
 +
</tr>
 +
<tr>
 +
<td>0ug/mL</td>
 +
<td>0mL</td>
 +
<td>5mL</td>
 +
</tr>
 +
</table><br><br>
 +
LAB Media (WCFS1) - prepare for Top 10
 +
<ul style="text-indent:30px;">
 +
<li>Added 5mL MRS into 3 test tubes</li>
 +
<li>Inoculate WCFS</li>
 +
<li>Incubate in 37C overnight</li>
 +
</ul><br><br>
 +
Followed plasmid purification protocol on red 1 and green 1 plasmid
 +
<ul style="text-indent:30px;">
 +
<li>250uL of each was used</li>
 +
<li>Optional 2nd wash with DNA buffer was done</li>
 +
<li>Stored in -20C</li>
 +
</ul><br><br>
 +
Made M9 media
 +
<ul style="text-indent:30px;">
 +
<li>63.2mL of H2O + .4g NH4Cl + .2g NaCl + 1.2g KH2PO4 + 2.7g Na2HPO4 + 16mL 5x M9 salt/H2O solution + 160uL MgSO4 + 8uL CaCl2 + .8mL 40% glucose (syringe filtering)</li>
 +
<ul><br><br>
</div>
</div>
</html>
</html>

Revision as of 00:53, 18 October 2014


Monday 9th June

Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2O

Autoclaved 1.5 mL Eppendorf tubes as well as LB

Poured plates of LB and chloramphenicol (CAM)

Streaked 2 plates with E. coli DguaB (pDCAF3)

Incubated in 37 degrees celsius

Aliquoted kanamycin (KAN) in four 1mL tubes

Tuesday 10th June

Autoclaved at 1:30pm

  • 0.8 grams of YNB + 156 mL H2O
  • 28 grams of salts + 490 mL H2O

Used sterile filter for 1M MgSO4 and 1M CaCl2

Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli

Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture

  • Labeled this "M9 media"

Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media

Placed sample in 30 degree celsius shaker for culturing

Wednesday 11th June

Inoculated CBB1 frozen stock in 5 mL of M9 media

Placed sample in 30 degree celsius shaker

Purified pDCAF plasmid using DNA mini kit

  • Plasmid concentration was 132.9 ng/uL


Friday 13th June

Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)

Followed bacterial transformation protocol to prepare/grow culture overnight

Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days

  • Previous CBB1 culture did not grow, showed slight growth
  • pDCAF3 strain in M9 did grow in the 30 degree celsius shaker


Monday 16th June

No growth in M9 by CBB1

To grow the promoter plasmid:

  • 5 mL of LB + 1 uL Amp
  • Label tubes
  • Grow in 30 degree celsius shaker overnight
New CBB1 trials:

  • 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample


Purify genomic DNA plasmid following the Wizard Protocol

  • Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
  • We put our 2 promoter plasmid plates in the 4 degree celsius shelf


Tuesday 17th June

Quantified gDNA with TECAN
  • Result: 5.4 ng/uL


Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker

Wednesday 18th June

Autoclaved 50% glycerol

Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):

  • CBB1 in M9 (x2 1.5 mL tubes)
  • Promoter 1
  • Promoter 2 (both promoter plasmids are J23114 Ampᴿ)
Purified both promoter plasmids using the purification protocol
  • Stored in the -20 degree celsius freezer in the DNA box


Thursday 19th June

Quantified promoter plasmid (J23114 #1 and #2)
    #1: 342.9 ng/ul with a ratio of 1.9 #2: 373.2 ng/ul with a ratio of 1.91


Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works
  • We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
  • Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
  • Then we will run gel electrophoresis: Concluded that the enzyme works


Cutting the promoter with restriction enzymes
  • Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Cutting linearized backbone
  • Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
  • Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius


Wednesday 9th July

Made 400mL of M9 solution
  • Made 40% glucose solution of 50mL: 2g glucose
  • Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4
  • Added 80mL 5x M9 salt solution (20% of 400mL)
  • Added 800uL MgSO4 + 40uL CaCl2
  • Added 4mL glucose solution using syringe
  • Stirred until all dissolved


Removed competent cells from the -80 freezer

Thawed on ice for 30 minutes

Removed CAM agar plate from 4 degree cold room

Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes

Placed mixture in water bath (42C) for 45 seconds

Placed mixture on ice for 2 minutes

Added 300uL LB to mixture

Placed in 37 degree shaker for 45 minutes

Plated cells on CAM plate, placed in 37 degree drawer overnight

Thursday 10th July

GuaB/pDCAF suspension in caffeine/theobromine
  • 0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM
  • 2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine
  • Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes
  • Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips
  • Place them into 37C inclubator overnight


Tuesday 15th July

Retrieved lactobacillus WCFS from Dr. Lu

Wednesday 16th July

Incubate 4 tubes with LB+CM
  • Red 1 and 2
  • Green 1 and 2
  • Placed in shaker at 12:20pm


Thursday 17th July

Made 1mg/mL Erm of 50mL

Used syringe to filter out

25mL Erm into LB agar

Made 13 places

Erm is in -20C

Friday 18th July

Streak out Erm-R onto Erm plates (made 2)

Put them into incubator (start: 9:35am)

Monday 21th July

cdh PCR redo:
  • Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O


Master Mix 1: Regular
  • 20uL Buffer G
  • 4uL forward primer
  • 4uL reverse primer
  • 0.4uL Q5 polymerase
  • 2uL template
  • 9.6uL H2O


Master Mix 2: Forward primer
  • 20uL Buffer G
  • 8uL forward primer
  • 0uL reverse primer
  • 0.4uL Q% polymerase
  • 2uL template
  • 9.6uL H2O


Master Mix 3: Reverse primer
  • 20uL Buffer G
  • 0uL forward primer
  • 8uL reverse primer
  • 0.4uL Q5
  • 2uL template
  • 9.6uL H2O


Master Mix 4: gDNA (10x)
  • 20uL Buffer G
  • 4uL forward primer
  • 4uL reverse primer
  • 0.4uL Q5 polymerase
  • 2uL template
  • 9.6uL H2O


PCR Procedure: Made 4 10uL samples of each master mix Set up thermocycler with the following settings:
  • 98C: 30 min
  • 40x cycles that ended up being:
  • 98C for 20 minutes
  • Gradient temperature for 20 minutes: 44.9C, 50.8C. 58.7C, 65.1C
  • 72C for 80 min
  • 72C for 5 minutes
  • Paused at 4C


Optimized shuttle vector growth environments: From 1 mg/mL concentrate
  • 1mL of each concentration into 2 falcon tubes
  • Inoculated 3mL LB with cells + Erm stocks
  • Also had 0ug/mL as a negative control
  • 8 tubes in total


Concentrations(ug/mL) H2O(mL) Erm(mL)
50 3.8 0.2
25 1.5 1.5
10 0.5 0.5
Inoculating Optogene Liquid Cultures:
  • Added 10mL liquid LB and 10uL chloramphenicol to each of the four falcon tubes
  • Picked colonies off each labeled plate and added to tubes as follows:
    • Plate
      • Green light 1
      • Green light 2
      • Red light sensor 1
      • Red light sensor 2
  • Incubated all 4 tubes in 37C shaker at 5:15pm


Inoculated E. coli guaB pdCAF (I4) in 3uL LB + 3uL CM

Inoculated Top 10 pdCAF (I5) in 3uL LB + 3uL CM

Placed in 37C shaker at 5:15pm

Tuesday 22th July

Made 80uL M9 + glucose solution
    Previous solution was contaminated 16uL 5x M9 salts 63.2uL H2O .4g NH4Cl, .2g NaCl, 1.2g KH2PO4, 2.712g Na2HPO4 16uL MgSO4 + uL CaCl2 .8mL 40% glucose solution using syringe Stir until dissolved


Erm concentration gradient
    Inoculated shuttle vector and top 10 cells Incubated in 37C


Erm LB
100ug/mL 0.5mL 4.5mL
50ug/mL 0.25mL 4.75mL
25ug/mL 0.125mL 4.875mL
0ug/mL 0mL 5mL


LAB Media (WCFS1) - prepare for Top 10
  • Added 5mL MRS into 3 test tubes
  • Inoculate WCFS
  • Incubate in 37C overnight


Followed plasmid purification protocol on red 1 and green 1 plasmid
  • 250uL of each was used
  • Optional 2nd wash with DNA buffer was done
  • Stored in -20C


Made M9 media
  • 63.2mL of H2O + .4g NH4Cl + .2g NaCl + 1.2g KH2PO4 + 2.7g Na2HPO4 + 16mL 5x M9 salt/H2O solution + 160uL MgSO4 + 8uL CaCl2 + .8mL 40% glucose (syringe filtering)


Wednessday 23th July



    50M EDTA solution
  • 0.166g of EDTA
  • pH at 8.3


    • Plasmid purification of shuttle vector -> 16.6 ng/uL

    Primers diluted + converted to 5uL stocks
  • Pro.bb rev
  • pdCAF3.bb for
  • Pro pdCAF3 for
  • BB.pdCAF3 rev


    • M9, caffeine, theobromine for I4/I5
  • Transferred 1mL of I4/I5 LB cultures into microcentrifuge tubes
  • Centrifuged, poured out supernatant
  • Resuspended in M9 + glucose
  • Diluted 50uM caffeine slution and 50uM theobromine solution down to 2000uM solutions, sterile filtered
  • Created 6 total culture tubes/placed in 37C shaker
  • 5uL I4 + 5uL M9+glucose
  • 5uL I4 + 3.75uL M9+glucose+1.25uL 2000 uM caffeine
  • 5uL I4 + 3.75uL M9+glucose + 1.25 uL 2000 uM theobromine
  • 5uL I5 + 5uL M9+glucose
  • 5uL I5 + 3.75 uL M9+glucose + 1.25uL 2000 uM caffeine
  • 5uL I5 + 3.75uL M9+glucose + 1.25uL 2000 uM theobromine


    • Placed 2 tubes in PCR cycler labeled ‘P’ and ‘S’
    Tube P
  • 30uL H2O
  • 10uL Phusion HF
  • 1uL dNTP
  • 2.5uL BB.pDCAF3 rev primer
  • 2.5uL pro.pDCAF3 for primer
  • 3.5uL pDCAF3 template
  • 0.5uL 5x Phusion DNA Polymerase

    • Tube S - Shuttle vector
  • 6uL H2O
  • 2uL Phusion HF
  • 0.2uL dNTP
  • 0.5uL pro.BB rev primer
  • 0.5uL pDCAF3.BB for primer
  • 0.7uL shuttle vector template
  • 0.1uL 5x Phusion DNA polymerase


    • Redid plasmid purification protocol on Red 1 and Green 1
  • Entire falcon tube was used
  • Optional 2nd wash with DNA wash was done
  • DNA is in -20C

Wednessday 23th July



    50M EDTA solution
  • 0.166g of EDTA
  • pH at 8.3


    • Plasmid purification of shuttle vector -> 16.6 ng/uL

    Primers diluted + converted to 5uL stocks
  • Pro.bb rev
  • pdCAF3.bb for
  • Pro pdCAF3 for
  • BB.pdCAF3 rev


    • M9, caffeine, theobromine for I4/I5
  • Transferred 1mL of I4/I5 LB cultures into microcentrifuge tubes
  • Centrifuged, poured out supernatant
  • Resuspended in M9 + glucose
  • Diluted 50uM caffeine slution and 50uM theobromine solution down to 2000uM solutions, sterile filtered
  • Created 6 total culture tubes/placed in 37C shaker
  • 5uL I4 + 5uL M9+glucose
  • 5uL I4 + 3.75uL M9+glucose+1.25uL 2000 uM caffeine
  • 5uL I4 + 3.75uL M9+glucose + 1.25 uL 2000 uM theobromine
  • 5uL I5 + 5uL M9+glucose
  • 5uL I5 + 3.75 uL M9+glucose + 1.25uL 2000 uM caffeine
  • 5uL I5 + 3.75uL M9+glucose + 1.25uL 2000 uM theobromine


    • Placed 2 tubes in PCR cycler labeled ‘P’ and ‘S’
    Tube P
  • 30uL H2O
  • 10uL Phusion HF
  • 1uL dNTP
  • 2.5uL BB.pDCAF3 rev primer
  • 2.5uL pro.pDCAF3 for primer
  • 3.5uL pDCAF3 template
  • 0.5uL 5x Phusion DNA Polymerase

    • Tube S - Shuttle vector
  • 6uL H2O
  • 2uL Phusion HF
  • 0.2uL dNTP
  • 0.5uL pro.BB rev primer
  • 0.5uL pDCAF3.BB for primer
  • 0.7uL shuttle vector template
  • 0.1uL 5x Phusion DNA polymerase


    • Redid plasmid purification protocol on Red 1 and Green 1
  • Entire falcon tube was used
  • Optional 2nd wash with DNA wash was done
  • DNA is in -20C


Thursday 24th July



    Ran gel for shuttle vector PCR, pDCAF3 PCR, Green 1, Red 1

    Created new Erm stock at 25mL and 1mg/mL
  • 25mL 100% ethanol
  • 25mg Erm


    • Divided LacBa competent cells into 24 PCR tubes, most having 90uL
  • Stored in -20C


    • E. coli strain media with Erm
    BL2
  • 2mL LB + 20uL BL2 + 0uL Erm
  • 2mL LB + 20uL BL2 + 50uL Erm
  • 2mL LB + 50uL BL2 + 100uL Erm

    • DH5
  • 2mL LB + 20uL DH + 0uL Erm
  • 2mL LB + 20uL DH + 50uL Erm
  • 2mL LB + 20uL DH + 100uL Erm


    • Glycerol stock of BL2/DH5
  • 3 glycerol stocks each strain
  • 500uL glycerol + 500uL strains


    • M9 Caffeine, I4/5
  • Took 1mL of I4/I5 LB cultures into microcentrifuge tubes
  • Spun for 2 min at 8000 rpm
  • Removed supernatant
  • Added 1mL of M9 media, resuspended
  • Spun for 2 min at 8000 rpm
  • Removed supernatant
  • Resuspended in 1mL of M9 media
  • Total of 4 culture tubes, put in 37C
  • I4 (50uL) +M9 (5mL) +CM34 (5uL)
  • I4 (50uL) + M9 (5mL) + Caffeine (500uL) + CM34 (5uL)
  • I5 (50uL) + M9 (5mL) + CM34 (5uL)
  • I5 (50uL) + M9 (5mL) + Caffeine (500uL) + CM34 (5uL)


    • Shuttle vector media
  • 4mL LB + 200uL Erm (1mg/mL)
  • Inoculate shuttle vector from agar stab
  • Incubate it on 37C shaker for 2-3 hours
  • 4 gradient test tubes
  • Incubate overnight at 37C


    • Final Conc.(ug/mL) Media(mL) LB(mL) Erm(uL)
    12.5 0.5 1.5 0
    65 0.5 1.5 46.7
    80 0.5 1.5 60
    100 0.5 1.5 77.8

Monday 28th July

Diluted caffeine and theobromine solutions for HPLC
  • 500uM Caffeine (10mL)
  • 500uM Theobromine (10mL)
  • 100uM Caffeine (10mL)
  • 100uM Theobromine (10mL)
  • 250uM Caffeine + 250uM Theobromine (10mL)


(page 169 on bottom, can’t read XW’s part)
  • Take 1mL of I4/I5 LB cultures and spin for 2 minutes at 8000 rpm
  • Removed supernatant
  • Added 1mL of OH salt, resuspended
  • 2 minutes at 8000 rpm
  • Removed supernatant
  • Resuspended in 1mL of M9 media

Wednesday 30th July

Ran gDNA extraction
  • Quantification: 1.8 ratio

Thursday 31th July

gDNA qualtify: 97.5 ng/mL, 1.76 ratio

cdh PCR
  • 30uL H20
  • 10uL Phusion HF
  • 1uL dNTP 10x
  • 2.5uL rev.
  • 2.5uL for.
  • 3.5uL template
  • 0.5uL polymerase
  • Made 4 10uL samples from master mix
  • Gradient: 44.9, 50.8, 58.9, 65.1C
  • Cycle 3 was used


    • Performed restriction digestion of pdCAF3 and green optogene
  • pdCAF3
    • .5uL Cutsmart buffer
    • .5uL Saci
    • .5uL EcoRI-HF
    • 7.52uL pdCAF3
    • 36.48uL H2O
  • Green
    • .5uL Cutsmart buffer .5uL NcoI .5uL NheI 8.4uL Green optogene 35.1 uL H2O
  • Incubated at 37C waterbath for 1 minute
  • Gel run at 120V for 25 minutes with a 1kb ladder


    • Cultured IG4/IG5 in M9 and no CM
  • 3mL M9, 300uL Caffeine, for only IG4, 30uL cells
  • Placed in 37 degree shaker

Monday 4th August

M9 Caffeine Media
  • 200uM 200mL caffeine
  • 50 M9 5x salts
  • .5mL MgSO4
  • .025mL CaCl2
  • 1.25mL 40% glucose


    • M9-Theobromine Media
  • 200mL 200uM Theobromine
  • 50mL M9 5x salts
  • .5mL MgSO4
  • .025mL CaCl2
  • 1.25mL 40% glucose


    • pdCAF3 Digestion
  • 5uL cutsmart buffer
  • 1uL PstI
  • .5uL XhoI
  • 7.52uL pdCAF3
  • 35.98uL H2O
  • 1 hour 37C incubation
  • 20 min 80C heat inactivation


Tuesday 5th August



    Ran gel of pdCAF3 digestion

    Set up feeding experiment cultures
  • Added 50uL caffeine M9/theobromine M9 to 250mL flasks
  • Added 50uL of CM34 to 4 of 6 flasks
  • Added 500uL of cells to flasks
  • Took 1mL of each culture as zero time point, placed in -20C
  • Placed flasks in 30C shaker

Wednesday 6th August



    Digsted pdCAF3 using XhoI
  • 5uL cutsmart buffer
  • .5uL XhoI
  • 7.52uL pdCAF3
  • 36.98uL H2O
  • 1 hour 37C incubation
  • Ran gel


    • Took 24 hour time point for feeding experiment, stored in -20

Thursday 7th August



    Took 48 hour time points for feeding experiments, stored in -20C

Friday 8th August



    Made 250uL 100uM (7.5pH) KPi buffer
  • 20.75uL of 1M K2HPO4
  • 4.25uL of 1M KH2PO4
  • 2.25uL of H2O


    • Centrifuged, washed experimental cultures of IG4 with KPi buffer

    Resuspended in 25uL KPi buffer

    Made 10% casamino acid solution
  • 5g casamino acids + 50mL H2O


Monday 11th August

    Resuspended primers in water
  • Spin down for 5 seconds
  • Inverted several times, set for 3 minutes
  • Inverte again, let sit for 3 more minutes
  • Invert before use


    • PCR
  • 57uL H2O + 1.6uL dNTP + 16uL buffer + 0.8uL polymerase in tube 1
  • 9uL of 1 + 0.25uL BB primer for + 0.25uL primer rev + 0.5uL template (PSBC13) in tube 8
  • Add 0.5uL pdCAF3 to tube 1
  • 9uL of 1 to tube 2-7, 0.25 rev + 0.25 for to each of the tubes respectively
  • Spin down for 5 seconds
  • Put tube 2-8 in PCR machine


Tuesday 12th August

    456uL H2O, 128uL buffer, 12.8uL dNTP, 6.4uL polymerase into tube A

    9uL of A + 0.25uL BB for + 0.25uL BB rev + 0.5uL purified green in tube 8

    0.5uL pdCAF to tube A

    72uL A to tube 1-6, 9uL A to tube 7, 0.25 BB rev/for to 7, 2uL for/2uL rev to 1-6 respectively

    Spin down for 5 seconds

    PCR in cycle 1
    Tube Primers Template Volume(uL)
    1 ndmA 1 pdCAF 80
    2 ndmA 2 pdCAF 80
    3 ndmB 1 pdCAF 80
    4 ndmB 2 pdCAF 80
    5 ndmC pdCAF 80
    6 ndmD + gst9 pdCAF 80
    7 BB pdCAF 10
    8 BB Green Optogene 10

Wednesday 13th August



    PCR
  • Add 114uL H2O, 32uL buffer, 3.2uL dNTP, 1.6uL polymerase to tube 1
  • 72uL of A to tube 10 + 20uL BB for + 2uL BB rev + 0.5uL purified green
  • 72uL of A to tube A + 2uL ndmD + gst9 + 2uL for/rev
  • Spin for 5 seconds
  • Put in PCR machine


    • Gel Purification
  • Cut one gel piece as small as possible
  • Add 500uL of buffer QG to tube 1-4, 10, 11, incubate at 50C for 10 minutes - invert several times until dissolved
  • Load liquid in column, spin 1 minute at full speed
  • Discard flow through, add 500uL QG, spin 1 minute at full speed
  • Discard flow through, add 750uL DNA wash buffer, spin 1 minute at full speed
  • Discard flow through, spin 1 minute at full speed
  • Discard flow through, add 40uL H2O, move column to fresh tube, spin 1 minute at full speed, sit for 1 minute
    • Tube ng/uL H2O(uL) uL
    ndmA 1 55.8 N/A 0,448
    ndmA 2 64 14 0.55
    ndmB 1 37.9 12 0.79
    ndmB 2 55.4 4 0.9
    ndmC 41.9 N/A 0.6
    ndmD/9 85.6 N/A 0.6
    BB 2.5 20 0.76

Thursday 14th August



    Ran gel

    Transformation
  • Throw Top 10 cells on ice for 10-20 minutes
  • Add 5uL of GG rxn mix then stir it gently on pipette
  • Leave cells on ice for 20-30 minutes
  • Heat shock at 42C for 5 minutes
  • Put 1mL LB into the tube, mix it, then transfer it and leave it in 37C for 1 hour
  • Plate 20uL and 200uL on LB+CM

</html>

Friday 15th August



    Pick 1-8 big colony
  • 4-10 middle colony
  • 17-24 small colony to 50uL LB


    • Set up 24 10uL PCR reactions
  • 260uL PCR
  • 52mL buffer
  • 5.2uL dNTPs
  • 0.5uL for ndmB
  • 1.5uL rev ndmB
  • 2.5uL polymerase
  • 180uL H2O


Monday 18th August



    Inoculate 23 and 24 (PCR) in 5mL LB

    Inoculate IG4/IG5 in 5mL LB


Tuesday 19th August



    Purification
  • Centrifuge 3200g for 10 min
  • Discard the culture media
  • Add 250uL solution 2/RNAse A, pipet up and down to mix thoroughly
  • Transfer suspension into a new 1.5mL microcentrifuge
  • Add 250uL solution II, invert until a clear lysate appears
  • Add 350uL solution II, then immediately invert several times until a white precipitate forms
  • Centrifuge at maximum speed for 10 minutes
  • Insert a mini column tube into a 2mL collection tube
  • Transfer the cleared supernatant to the column
  • Centrifuge at maximum speed for a minute
  • Discard the filtrate and reuse the collection tube
  • Add 50uL HBC buffer
  • Centrifuge at maximum speed for one minute
  • Discard the filtrate and reuse collection tube
  • Add 700uL DNA wash buffer
  • Centrifuge at maximum speed for 1 minute
  • Discard the filtrate and reuse the collection tube
  • Centrifuge the column for two minutes at maximum speed to dry the column matrix
  • Transfer the mini column to a clean 1.5mL microcentrifuge tube
  • Add 60uL elution buffer to the column
  • Sit at room temp for 1 minute
  • Centrifuge at maximum speed for 1 minute
  • Store DNA at -20C


    • Wash cells
  • 1mL to fresh tube
  • 2.5 minutes at 8000g
  • Remove all but the pellet, resuspend the salt
  • 2mL salt + 8mL H2O


    • Make M9
  • 500uL 40% glucose
  • 100uL MgSO4
  • 5uL CM
  • 10mL 5x salts
  • 1mL casamino acids
  • 34mL H2O
  • Make 9 test tubes, 3 with IG4, 3 with IG5, and 3 with IG4, each having caffeine, theobromine, or water

Thursday 21th August



    Digestion
  • 1uL cutsmart buffer
  • 1uL Xho
  • 8uL DNA

Monday 25th August



    240uL
  • 120uL Buffer G
  • 105uL H2O


    • PCR
  • Backbone: 80uL x2 (purified green and associated primers)
  • cdhA: 40uL (CBB1 with Xba + cdhBA)
  • cdhBC: 40uL (CBB1 with Nsi + cdhAB)


    • Feeding assay
  • Inoculated Top 10, IG4, and IG5 in LB
  • Autoclaved 3 250mL flasks
  • Ran gel of iGEM BB PCR


    • Inoculation
  • Inoculated E. coli in 3uL LB + 3uL CM
  • 37C shaker

Tuesday 26th August



    Purifying the shuttle vector plasmid (yielded 34.3 ng/mL, 1.83)
  • Use purification protocol from 8/19


    • Electroporated plasmid into lactobacillus (shuttle vector)
  • 2-mm cuvette, 1.7kV, ~5ms, ~200uL competent cells, 20uL plasmid DNA
  • Recovered in 1900uL MRS medium, incubated for 2.5 hours at 37C
  • Plated on various CM MRS plates at 30C (25uL Lactobacillus shuttle vector cells)


    • Made CM-MRS plates
  • 1 plate with 0 CM
  • 1 plate with 5ug/mL CM
  • 2 plates with 10ug/mL CM
  • 2 plates with 20ug/mL CM


    • Inoculated IG4, IG5, Top 10
  • 5mL LB
  • IG4/5 inoculations involved 5uL of CM solution (1000x dilution)
  • Placed in 37C

Wendesday 27th August



    Prepared the following:
  • 10mL 1x M9 salts (2mL 5x + 8mL H2O)
  • 40mL concentrated M9 + 1% casamino acids
  • 50mL 2mM caffeine solution (58mg caffeine + 50mL H2O)
  • 50mL 2mM theobromine solution (54mg theobromine + 50mL H2O)


    • Washed overnight cultures using 1x M9 salts

    Prepared feeding assay as follows (in chart)

    Removed 500uL of each culture, labeled with date and time 0, in -20C

    Cultures placed in 37C
    Tube1 Tube2 Tube3 Tube4 Tube5 Tube6 Tube7 Tube8 Tube9
    Conc. M9+CA(mL) 4 4 4 4 4 4 4 4 4
    2mM Caff(mL) N/A N/A 1 N/A N/A 1 N/A N/A 1
    2mM Theo(mL) N/A 1 N/A N/A 1 N/A N/A 1 N/A
    H2O(mL) 1 N/A N/A 1 N/A N/A 1 N/A N/A
    Cells(50uL) Top 10 Top 10 Top 10 Top 10 Top 10 Top 10 Top 10 Top 10 Top 10
    CM34(uL) N/A N/A N/A 5 5 5 5 5 5


    160mL M9 + 1% casamino acids
  • 100mL H2O
  • 20mL 10% casamino acids
  • 20uL 1M cach(?)
  • 400uL 1M MgSO4
  • 40mL 5 M9 salts


    • 10mM caffeine
  • 290mg caffeine + 50mL H2O
The Institute for Genomic Biology
Biosystems Design, South wing, 2nd floor
University of Illinois at Urbana-Champaign
1206 West Gregory Drive
61801 Urbana, Illinois
illinoisigem@gmail.com
Copyright (c) 2014 igem.org. All rights reserved.

Retrieved from "http://2014.igem.org/Team:UIUC_Illinois/Notebook"