AlanFung:LabNotes/CTCF/2011-7-11

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Identify source of loss during Nextera Bisulfite conversion with 10ng gDNA[edit]

Objective[edit]

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

Samples[edit]

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[edit]

Low-Input Tagmentation with Nextera[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

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

Amplification with Kapa and Klenow[edit]

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[edit]

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[edit]

  • 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, new tips)
  • Repeat experiment with 1ng gDNA
  • Do not add extra water to #2