AlanFung:LabNotes/CTCF/2011-9-20
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Notes[edit]
- Instead of using protease and NP40 lysis buffer, we will use NaOH for cell lysing, ideally NaOH should be made fresh as it will absorb carbon dioxide and form sodium bicarbonate which does not lyse cells
- Initially I will repeat the experiment with both lysis methods, and run a PCR analysis (this will tell us which method is better and also tell us wether bisulfite conversion works)
- In the original imprint protocol, we adjust the total volume of DNA sample to 24ul with water and add 1ul NaOH (Balance Solution), vortex and incubate sample at 37C for 10min, in order to denature the DNA samples
- Protocol for cell lyses found online uses 200mM NaOH/1%SDS (Where the NaOH breaks down the cell wall and denatures the DNA, while SDS helps by making the cell wall soluble and denatures the protein)
- The question now is what is the concentration and volume needed to lyse the cells, we have no information about the concentration of the provided NaOH, I will try to use it for cell lyses with 1% of SDS.
- Prepare Stock Balance Solution with 1% SDS
- After adding NaOH gentle mixing is required so the DNA is not damaged. The DNA will have various (relatively large) proteins bound and anything more than gentle inversion or rolling the falcon can shear the DNA
Prepare 10% SDS[edit]
- Add 1g of SDS in 10ml of solvent
- Dilute 10% SDS to 1% SDS
- Add 0.1ul 10% SDS to 0.9ul Balance solution (NaOH)
Cell Lysis[edit]
- I would like to do an alkaline cell lysis with NaOH, after doing some research the required amount of OH- has to be at least 20mM NaOH in PBS (pH 11.7) for quick cell lysis
- We will test the pH of the NaOH with litmus paper Range 0-14 (Ordered placed on 9/20)
- Add 1ul NaOH to 24ul nuclease free water, test pH with litmus paper (Increase amount of NaOH if the pH is not high enough)
- Both pH are at close to 14
- Seems like the NaOH from the kit is basic enough to lyse the cells
NAOH
- Add 23 uL 0.5 mg/mL BSA to cells and
- Add 1 uL Imprint Balance solution to each sample
- Incubate at 37 C for 10 minutes
- Cells are now lysed
NP40 w Protease
- Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
- Add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells;
- incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes
- Cells are now lysed
- Add 21 uL 0.5 mg/mL BSA and
- Add 1 uL Imprint Balance solution to sample
- incubate at 37 C for 10 minutes
Bisulfite Conversion - Imprint Kit
- Prepare DNA Modification Solution (CT Conversion Reagent):
- Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes
- Perform Column Purification of each sample:
- Elute into 11uL Elution Buffer
qPCR w bisulfite identification primers
Layout
NP40-8 |
NP40-21 |
NP40-22 |
NaOH-8 |
NaOH-21 |
NaOH-22 |
- Prepare Master Mix
MM | CHR8 | CHR21 | CHR22 |
2X iQ Super Mix | 44 | 44 | 44 |
Primer F (3.3uM) | 13.2 | 13.2 | 13.2 |
Primer R (3.3uM) | 13.2 | 13.2 | 13.2 |
gDNA | Do not add to MM | Do not add to MM | Do not add to MM |
H2O | 13.2 | 13.2 | 13.2 |
- Add 2ul of the modified DNA to a qPCR striptube, add 39ul MM to each tube
Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever