Identify source of loss during Nextera Bisulfite conversion with 10ng gDNA[edit]
Objective[edit]
- Want to figure out the source of loss during Nextera Bisulfite conversion
- 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
|
- Nextera Low-Input, Normal Protocol
- Nextera Low-Input, Heat
- Nextera Low-Input, Heat, Column Purification
- Nextera Low-Input, Heat, Desulphonate, and Column Purify
- Nextera Low-Input, Heat, Bisulfite, Desulphonate, and Column Purify
- 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
- 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