AlanFung:LabNotes/CTCF/2011-5-10
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Bisulfite Conversion DNA Damage Estimation
- Perform PCR with CHR8, CHR21 and CHR22
- These will be our standards
File:ZhangLab 2 2011-05-10 10hr 40min.jpg
- Need to purify CHR8, CHR21, and CHR22.
- Qubit Results(ng/ul)
- Assume amplicon length ~200bp
- CHR8-3.25ng/ul 26.72nM
- CHR21-4.39ng/ul 36.09nM
- CHR22-4.77ng/ul 39.21nM
- It is better to perform a size selection, since there are some ghosting on the gel
Size Selection
- Prepare 3 gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
- Cut out the correct band (~200 nt in this case), transfer gel slice to shearing assemblies
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- Spin down all tubes at 13,000rpm for 2min
- In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
- Perform ethanol precipitation
Qubit Quantifiction
- CHR8-
- CHR21-
- CHR22-
Determine the efficiency of bisulfite conversion
- After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of converted human genomic DNA. Base on the we can determine the conversion efficiency.
Make Serial Dilution on Control DNA
- Create 6 STDs
Sample Name | dsDNA Concentration (pM) |
Std 1 | 20 |
Std 2 | 2 |
Std 3 | 0.2 |
Std 4 | 0.02 |
Std 5 | 0.002 |
Std 6 | 0.0002 |
Prepare qPCR Reagents
Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing
- 2X KAPA SYBR FAST qPCR Master Mix
- 6 DNA Standards
- 1:1000 dilution of bisulfite converted library DNA
- Primer Mix (F+R) 1:1 ratio 1.65uM
Prepare qPCR
CHR8 | CHR21 | CHR22 | |
2X iQ Super Mix | 20 | 20 | 20 |
Primer F+R (1.65uM) | 4.85 | 4.85 | 4.85 |
gDNA | 8 | 8 | 8 |
ddH2O | 7.15 | 7.15 | 7.15 |
Total | 40 | 40 | 40 |
Perform real-time PCR reaction in thermocycler
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
Generate Standard Curve
For Bio-Rad Real time Thermal Cycler
- Edit plate setup
- Select and add wells with control DNA as Standard
- Specify Quant Standards (bottom right button)
- After PCR cycle
- Select Quantitation Tab on left column to see standard curve
- Select Calculations Tab at the bottom to see the sample concentrations
For Eppedorf Real time Thermal Cycler
- Edit Plate Layout
- Select wells for standards
- right click on well and select well as standard
- Input name and standard amount
- After PCR cycle
- Standard curve will show up on the bottom half of the analysis screen
- Sample concentrations will show up on left column