AlanFung:LabNotes/CTCF/2011-5-10

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Revision as of 00:40, 26 May 2011 by >Alan6017518 (→‎Size Selection)
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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.

Qubit Quantifiction

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

Generate Recovery Curve