Template:Heidelberg/notebook/pc/week26
week number 26
▼2015-06-22 E.coli Trafo with Plasmids form the iGEM registry
To build the protein construct the mTagBFP BBa_K592100 from the iGEM registry were used.
1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Do not remove the foil cover.
2. Pipette 10µl of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.
3. Thawing 50µl chemical competent E. coli on ice.
4. Add to 50µl competent cells:
1µl DNA from the registry
10µl KCM 5x
39µl H2O
5. Incubate on ice for 30 minutes
6. Heat shock at 42°C for 2 minutes
7. Incubate on ice for 2 minutes
8. Add 900 µl of LB or 2x YT Medium
9. Incubate on 37°C for 60min
10. Centrifuge 5min at 1000g
11. Take 900µl of supernatant and throw away
12. Resuspend pellet in remaining media
13. Plate out on agar with the appropriate antibiotic and grow overnight at 37°C
▼2015-06-24 QIAprep Spin Miniprep Kit
Miniprep with mTagBFP BBa-K592100 and the pUB23-S-ßGal plasmid was performed.
1. Prepare o/n culture
2. Perform mini prep according to manufacturer’s protocol
Durchführung
▼2015-06-25 E. coli glycerol stocks
Glycerol stocks from E.coli containing mTag BFP BBa_K592100 or pUB23-S-ßGal were made.
1. Grow up an overnight culture of strains of interest
2. Transfer 500µl into a safe lock reaction tube
3. Add 500µl of 40% sterile glycerol solution
4. Freeze slowly at -80°C
Durchführung
▼2015-06-25 Fragment amplification for protein construct (PCR)
To obtain the required Gibson Fragments (Backbone (p413-GPD), mTagBFP, Ubiquitin, sfGFP) PCRs were performed.
reaction mixture:
12.5µl Q5 High-Fidelity 2x Master Mix
0.5µl DNA
1.25µl fwd primer 10µM
1.25µl rev primer 10µM
9.5µl H2O
Program:
95°C 2min
95°C 30sec
63-66°C 30sec (mTagBFP: 63°C; Ub: 65°C; sfGFP: 64°C; p415-GDP: 66°C)
72°C 30sec
72°C 5min
35 cycles
gel:
TAE buffer
1% w/v agarose
loading samples:
5µl Ladder
25µl PCR sample
5µl Loading buffer
▼2015-06-27 Fragment amplification for protein construct (PCR) (continued)
Results
PCRs for Gibson fragments mTagBFP, Ubiquitin and sfGFP worked because a bands are visible with the expected size. No p415-GPD product was visible. Therefore the p415-GPD PCR need to be optimised.
Gel Extraction
mTagBFP, Ubiquitin and sfGFP fragments were separated in 1% agaros.
Gel extraction was performed according to QIAGEN QIAquick Gel Extraction Protocol. (Elution in 30µl EB)
▼2015-06-27 Phusion PCR with DMSO and GC Buffer
To amplify the p415-GPD plasmid with Gibson overhangs an optimized gradient PCR program was used.
reaction mixture:
total:10µl
5.0µl Phusion 2x Master Mix
0.2µl DNA
0.5µl xxJD007xx 10µM
0.5µl xxJD008xx10µM
0.5µl DMSO
1.3 H2O
programm:
(PHGRAD)
95°C 2:00min
95°C 0:30min
58°C-70°C 0:30min
72°C 3:00min
72°C 5:00
35 cycles
gel:
TAE buffer
1% w/v agarose
loading samples:
5µl PCR sample
2µl Loading buffer
Results
no product. PCR need to be repeated.
▼2015-06-27 p415-GPD test digest
To check if the right plasmid is in the tube a KpnI digest was performed.
Set up reaction according to protocol:
- ddH2O for a final volume of 20 µl
- 2 µl of 10x 1.1 Reaction Buffer
- 0.5 µl of KpnI
- 1 µl of DNA
Incubate at 37°C for 60'
Load on gel (add loading dye first)
Results
The gel shows the expected bands. The p415 plasmid is in the tube.