Difference between revisions of "Template:Heidelberg/notebook/cf/week30"
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| + | Figure 5: The second mutation PCR of the CFTR-testconstruct | ||
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Latest revision as of 16:52, 18 September 2015
week number 30
▼2015-07-24 Yeast transformation with p415-GPD with CFTR-testconstruct without mutation correction
Description
Yeast was transformated with p415-GPD with the CFTR-testconstruct from a miniprep of an E.coli culture. Transformed cells should turn red.
Procedures:
Yeast transformation 24.07
Description:
Materials and chemicals:
10 µl of cells for transformation with a plasmid, 50 µl of cells for transformation with a PCR product
2 µl of plasmid DNA per 10 µl of cells (200 ng of DNA)
6 equivalents of PEG to the yeast cells
1/9 of DMSO to the volume of plasmids, yeast cells and PEG
100 - 200 µl of liquid medium
Steps:
- Give the plasmid DNA into a sterile 1.5 ml tube (2 µl per 10 µl of cells. Add the thawed competent cells.
- Mix the suspension well, then add the PEG.
- Incubate for 30 mins at room temperature while mixing (the cells can be incubated up to 2 hours).
- Add the DMSO to get a final concentration of 10% DMSO.
- Place the yeast in a 42°C water bath for 5-20 minutes. NO Thermomixer.
- Centrifuge cells for 2-3 minutes at 2000 rpm/500g
- Discard the supernatant and resuspend the yeast in the liqiud YPD medium
The transformation was repeated on 27.07.2015
Results:
Transformation 1:
The first transformation resulted in cultures with an ununsual behaivior, maybe the plates were contaminated with another organism. The cells didn't turned red
Transformation 2:
A test run on a FACS showed that the transformed cells are functional and express the CFTR-testconstruct
▼2015-07-25 Mutation correction PCR p415-GPD with CFTR-testconstruct
Description
In order to correct the mutation in the mCherry part of the CFTR-testcontruct, a mutation PCR was made.
Procedures:
Mutation correction PCR 1:
Description
Chemicals:
0,1 µl p415-GPD with CFTR-testconstruct (about 650 ng/µl)
1 µl Primer fwd
1 µl Primer rev
2,9 µl ddH2O
5 µl Q5
Program:
2-step PCR:
98°C
-----------------------
98°C
72°C
------------------------
72°C
------------------------
4°C for holding
Mutation correction PCR 2
Description:
Chemicals:
0,1 µl p415-GPD with CFTR-testcontruct
1 µl Primer fwd
1 µl Primer rev
2,9 µl ddH2O
5 µl Q5
Program:
98°C for 1 minute
------------------------
98°C for 20 s
Anneal for 20 s
72°C for 4 minutes
Repeat cycle 35 times
-------------------------
72°C for 5 minutes
-------------------------
4°C for holding
Tried annealing temperatures:
72°C (2-step-PCR)
70,5°C
68,8°C
65,6°C
64°C
Annealing temperatures were made by gradient
Results:
Mutation PCR 1:
The Gel showed there was just smear
Mutation PCR 2:
Every Annealing temperature resulted in smear and no detectable fragment
