Template:Pitt/labnotebook2

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 Table 2:

Loading Buffer Concentration Table:

x= total loading volume

Lane

Sample

Amount Loaded (ul)

1

1kb DNA Ladder

2

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:

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

Reagent Table 1 (w/o RBS):

Reagent Table 2 (w/ RBS):

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 Table 2 (w/ RBS):

PCR Tube Labeling:

1 = fwd primer + rev primer RBS

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

New PCR Condition:

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: