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

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==Objective==
==Objective==
*test the feasibility of using zymo direct kit to perform bisulfite conversion on 100 cells, 200 cells and 1000 cells worth of DNA and perform 2nd strand synthesis to make sequencing library with regular nextera kit
*test the feasibility of using zymo direct kit to perform bisulfite conversion on 100 cells, 200 cells and 1000 cells worth of DNA and perform 2nd strand synthesis to make sequencing library with regular nextera kit
*Test wether we need to perform heat denaturing on the bisulfite converted library to ensure we have single stranded DNA after conversion with 3 set of samples.
==Protocol==
==Protocol==
*Setup 3 sets of reactions (Set 1, 2 & 3)
*Setup 3 sets of reactions (Set 1, 2 & 3)

Revision as of 18:00, 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

Objective

  • test the feasibility of using zymo direct kit to perform bisulfite conversion on 100 cells, 200 cells and 1000 cells worth of DNA and perform 2nd strand synthesis to make sequencing library with regular nextera kit
  • Test wether we need to perform heat denaturing on the bisulfite converted library to ensure we have single stranded DNA after conversion with 3 set of samples.

Protocol

  • 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
  • Transfer to qPCR tubes

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 Scale up
gDNA 3 12
1:50 Nextera illumina compatible transposomes 1 1
5X Nextera HMW Buffer 1 3
  • Incubate at 55C for 5min
  • Mix on ice
Content Volume per reaction
Nuclease Free Water 7.6
10uM Orange Primer 1
10uM Blue Primer 1
Bisulfite converted DNA w/ 2nd strand 15
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