AlanFung:LabNotes/CTCF/2011-6-7: Difference between revisions

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==Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA==
==Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA==
*Scale up volume accordingly (~12ul)
*Scale up volume accordingly (total volume ~12ul after 2nd strand synthesis)
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Revision as of 17:28, 9 June 2011

qPCR with CHR8 primer using pfu Cx polymerase on bisulfite converted GM12878 gDNA

Protocol

Component amount per reaction
distilled water 35.6
10x pfuturbo cx reaction buffer 5
dNTPs(10mM each DNTP) 1
DNA template 1
CHR8 F primer 3.3uM 3
CHR8 R primer 3.3um 3
sybr green 50x 0.4
pfuturbo cx hotstart dna polymerase 2.5U/ul 1
total reaction volume 50
    • Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m
95C 30s
52C 30s
72C 2m
Plate Read
Go to step 2 repeat for 29 times
72C 10m
15C hold

Results

File:060611 CHR8 pfuCx.jpg

  • Amplification failed with Pfu cx polymerase

Perform bisulfite conversion on 6ng, 1.2ng, and 600pg of gDNA using Zymo’s direct kit

  • Setup 3 sets of reactions (Set 1, 2 & 3)
Tube Amount Volume per Reaction Tube Concentration H2O
A 6ng 1.2 5ng/ul 18.8
B 1.2ng 6.82 176g/ul 13.18
C 600pg 3.41 176g/ul 16.59
D Blank 0 N/A 20
  • Warm CT conversion Reagent (prepared on 6/6/11) up to 37C and vortex in 37C incubator
  • Add 130ul CT Conversion Reagent to each tube
  • Elute with 11ul elution buffer

Synthesize 2nd strand with N9 primer and klenow exo

  • Set 1 denature at 95C
  • Set 2 denature at 84C
  • Set 3 no heat denaturing
  • Heat up to 95C, 84C and no heating for 7 mins and quickly transfer to cooling rack, wait for 1 min.
  • Add 1 uL Klenow Polymerase(1U/ul)
  • Add 0.52ul 5uM N9 Primers
  • Add 1ul dNTPs (10mM each DNTP)
  • Incubate for 10min at 30 C

Perform standard tagmentation and PCR amplification with orange/blue primers using KAPA

  • Scale up volume accordingly (total volume ~12ul after 2nd strand synthesis)
Content Volume per reaction
gDNA 3
1:50 Nextera illumina compatible transposomes 1
5X Nextera HMW Buffer 1
  • Incubate at 55C for 5min
  • Mix on ice
Content Volume per reaction
Nuclease Free Water 17.6
10uM Orange Primer 1
10uM Blue Primer 1
Bisulfite converted DNA 5
50X SYBR Green 0.4
Kapa Supermix 25
Total 50
    • Perform Manufacturer protocol for KAPA
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C
    • Cycle the samples in a thermocycler under the following conditions
  • 3. Purify the tagged DNA using qiaquick column
  • Page Quantification