AlanFung:LabNotes/CTCF/2011-5-10: Difference between revisions
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*Create 6 STDs | *Create 6 STDs | ||
{| {{table}} | {| {{table}} | ||
| Sample Name|| | | align="center" style="background:#f0f0f0;"|'''Sample Name''' | ||
| align="center" style="background:#f0f0f0;"|'''Concentration (pM)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''DNA Molecule''' | |||
|- | |- | ||
| | | STD1||20||19.44||9.01E+10 | ||
|- | |- | ||
| | | STD2||2||1.944||9.01E+09 | ||
|- | |- | ||
| | | STD3||0.2||0.1944||9.01E+08 | ||
|- | |- | ||
| | | STD4||0.02||0.01944||9.01E+07 | ||
|- | |- | ||
| | | STD5||0.002||0.001944||9.01E+06 | ||
|- | |- | ||
| | | STD6||0.0002||0.0001944||9.01E+05 | ||
| | |||
| | |||
|} | |} | ||
Revision as of 17:48, 1 June 2011
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
File:ZhangLab 2 2011-05-26 17hr 09min.jpg
- 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
- Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content | Volume Needed | Volume |
100% Ethanol | 2.5X Sample Volume | 950uL |
3M NaOAC | 0.1X Sample Volume | 38uL |
Glycoblue | 1/300X Sample Volume | 2uL |
Sample | Sample Volume | 380uL |
Total Volume | 1370uL |
- Mix all content by repeat pipetting using multichannel pipette
- Store in -80C for more than 20 mins or forever
- Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
- Centrifuge frozen 1.5mL tubes at 10000rpm for 30m at 4C
- Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
- Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
- Centrifuge at 10,000rpm for 5min at 4C
- Remove all liquid, air dry sample for 5min or until samples dried out
- Resuspend the DNA with 20uL RNAse free H2O
Qubit Quantifiction
- CHR8-1.87ng/ul
- CHR21-0.545ng/ul
- CHR22-1.12ng/ul
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 | Concentration (pM) | Amount (ng) | DNA Molecule |
STD1 | 20 | 19.44 | 9.01E+10 |
STD2 | 2 | 1.944 | 9.01E+09 |
STD3 | 0.2 | 0.1944 | 9.01E+08 |
STD4 | 0.02 | 0.01944 | 9.01E+07 |
STD5 | 0.002 | 0.001944 | 9.01E+06 |
STD6 | 0.0002 | 0.0001944 | 9.01E+05 |
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