AlanFung:LabNotes/CTCF/2011-7-11

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Revision as of 22:45, 12 July 2011 by >Alan6017518 (→‎To do)
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Identify source of loss during Nextera Bisulfite conversion with 10ng gDNA

Objective

  1. Want to figure out the source of loss during Nextera Bisulfite conversion
  2. Use qPCR to identify the source of the loss

Samples

Protocol 1 2 3 4 5 6 (NTC)
Low-Input Nextera X X X X X X
Protease Digestion (1:100 Dilution) X X X X X X
Klenow Extension X X X X X X
Heat X X X
Heat w/ Bisulfite X
Desulphonation X X
Column X X X
Amplify X X X X X X
  1. Nextera Low-Input, Normal Protocol
  2. Nextera Low-Input, Heat
  3. Nextera Low-Input, Heat, Column Purification
  4. Nextera Low-Input, Heat, Desulphonate, and Column Purify
  5. Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify
  6. NTC - normal protocol
  • "Nextera Low-Input": Perform Shendure Protocol, Protease Treat with 1/100 Dilution, Klenow Extend
    • Used 1 uL of 1 ng/uL Jurkat DNA in first five tubes, 1 uL H2O in NTC
  • Normal Protocol: Amplify using KapaKlenow Protocol
  • Heat: After heat, amplify using KapaKlenow Protocol
  • Heat/Column: After heat, purify through Zymo column, skipping desulphonation step; amplify using KapaKlenow Protocol
  • Heat/Desulphonate/Column: After heat, purify through Zymo column with desulphonation step; amplify using KapaKlenow Protocol
  • Heat/Bisulfite/Desulphonate/Column: After heating in bisulfite solution, purify through Zymo column with desulphonation step; amplify using KapaKlenow protocol
  • NTC: Just do normal protocol, but with water instead of 1 ng DNA

Protocol

Low-Input Tagmentation with Nextera

  • Add 1 uL of 1:50 Diluted Nextera Transposase, 1 uL Nextera 5x HMW Buffer, and 2 uL H2O to 0.2ul GM12878 gDNA (50ng/ul)
  • Incubate at 55 C for 5 minutes

Protease Digestion

  • Add 1 uL of 1:100 diluted Qiagen Protease (5 AU/mL, diluted to 0.05 AU/mL) to solution
  • Incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes

Klenow Extension

  • Add 1 uL of 10 U/uL Klenow exo-minus DNA Polymerase and 1 uL of 1 mM Nucleotide Mix (dATP, dGTP, m-dCTP, dTTP) to solution
  • Incubate at 30 C for 30 minutes

Bisulfite Conversion

  • Add 130 uL of CT Conversion Reagent (Zymo Bisulfite direct Kit) to solution
  • Incubate at 98 C for 8 min, 64 C for 3.5 hours; hold at 4 C when finished

Column Purification with/without Desulphonation

  • Follow instructions in Zymo Bisulfite Direct kit for purification
  • Skip desulphonation step when necessary

Amplification with Kapa and Klenow

Content Volume MM
DNA 10 Do not add to MM
H2O 12 79.2
10uM Orange 1 6.6
10uM Blue 1 6.6
10U/ul Klenow Exo- 1 6.6
2XKapa 25 165
total 50 330
  • KapaKlenow uses Klenow for 2nd strand synthesis, Kapa for PCR
  • Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL Kapa SYBR Supermix, and 1 uL Klenow exo-minus (10 U/uL) to solution
  • Perform PCR using the following thermocycler settings:
30 C, 10 minutes
72 C,  3 minutes
95 C, 30 seconds
==
95 C, 10 seconds
58 C, 30 seconds
72 C, 3 minutes
== x 40 cycles
72 C 10 minutes
Hold at 4 C

Results

File:Screen shot 2011-07-12 at 2.53.16 PM.jpg

  • Contamination is pretty bad, NTC got amplified first!
  • Shendure goes up with Shendure+heat, actually shendure+heat comes up faster than shendure alone, since heat is suppose to do some damage.
  • Heat+Desulphonation+column vs no desulphonation doesn't make sense either, for desulphonation is another DNA damaging step
  • heat vs heat+column shows that column does have an effect on sample loss
  • bisulfite step is the major player in terms of sample loss

To do

  • Repeat experiment with another NTC that goes through bisufite step
  • Eliminate contamination (Clean and UV PCR Hood, UV all tubes and, Use UV RT-PCR grade water)
  • Repeat experiment with 1ng gDNA
  • Do not add extra water to #2