AlanFung:LabNotes/CTCF/2011-8-26
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Low-input Bisulfite Conversion and MDA Nextera library
Protocols Today
- Start with four tubes: 314 cells, 157 cells x 2, NTC all go through bisulfite conversion
- Make 1571 cell/ul (original tube)
- Make 314 cells/ul (dilute 10ul cells in 40 ul ddh2o)
- Make 157cells/ul (dilute 10ul cells in 90 ul ddh2o)
Cell Lysis
- First, make protease Dilution
- Add 3 uL Protease to 97 uL H2O for a 3:100 dilution
- Next, 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
Make New BSA-Water solution
- Stock solution is at 10mg/ml, need to dilute to 0.5mg/ml
- Take 10ul stock BSA dilute with 190ul nuclease free water
Bisulfite Conversion - Imprint Kit
- Prepare DNA Modification Solution (CT Conversion Reagent):
- Add 21 uL 0.5 mg/mL BSA and
- Add1 uL Imprint Balance solution to each sample;
- incubate at 37 C for 10 minutes
- 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
Continue on 8/27
- Denature all samples at 95C for 30sec and quickly transfer to cooler rack