AlanFung:LabNotes/CTCF/2011-7-12
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Improved protocol for Identifying source of loss during Nextera Bisulfite conversion with 1ng 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
- Eliminate source of contamination
Samples[edit]
Protocol | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Low-Input Nextera | X | X | X | X | X | X | X |
Protease Digestion (1:100 Dilution) | X | X | X | X | X | X | X |
Klenow Extension | X | X | X | X | X | X | X |
Heat | X | X | X | ||||
Heat w bisulfite | X | X | |||||
Desulphonation | X | X | X | ||||
Column | X | X | X | X | |||
Amplify | X | X | X | X | X | X | X |
- Nextera Low-Input, Normal Shendure 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
- NTC1 - normal shendure protocol
- NTC2 -shendure + bisulfite 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
- NTC1: Just do normal protocol, but with water instead of 1 ng DNA
- NTC2: repeat shendure protocol and bisulfite conversion with NTC
Protocol[edit]
Elimination of source of contamination[edit]
- Clean with EtOH and DNAse wash hood and UV fo 15mins
- Use newly opened tips
- UV all tubes
- UV RT-PCR Grade water, EB Buffer and Protease
Low-Input Tagmentation with Nextera[edit]
- Add 1 uL of 1:50 Diluted Nextera Transposase, 1 uL Nextera 5x HMW Buffer, and 1 uL H2O to 2ul GM12878 gDNA (0.5ng/ul)
- Sample 1-5
Content | Volume | MM |
1:50 Nextera Transposase | 1 | 5 |
5X Nextera HMW Buffer | 1 | 5 |
RT-PCR Water | 1 | 5 |
0.5ng/ul gDNA | 2 | 10 |
Total | 5 | 25 |
- Sample 6-7
Content | Volume | MM |
1:50 Nextera Transposase | 1 | 5 |
5X Nextera HMW Buffer | 1 | 5 |
RT-PCR Water | 3 | 15 |
0.5ng/ul gDNA | 0 | 0 |
Total | 5 | 25 |
- 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
- Add 5ul to sample 1-5
- Add 2 ul to sample 6-7
- 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
- Add 5ul klenow and 5ul dNTP to sample 1-5
- Add 2 ul klenow and 2 ul dNTP to sample 6-7
- Incubate at 30 C for 30 minutes
- Aliquot 8ul to each tubes
- keep sample 1 & 6 at 4C for later amplification
Bisulfite Conversion[edit]
- Add 130 uL of CT Conversion Reagent (Zymo Bisulfite direct Kit) to sample 5 and 7
- Incubate at 98 C for 8 min, 64 C for 3.5 hours; hold at 4 C when finished
- Incubate sample 2, 3, and 4 at a separate block for it has less volume
Column Purification with/without Desulphonation[edit]
- 4, 5 and 7 desulphonation sample 3 no desulphonation
- Set sample 2 aside until amplification
- 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 | 92.4 |
10uM Orange | 1 | 7.7 |
10uM Blue | 1 | 7.7 |
10U/ul Klenow Exo - | 1 | 7.7 |
2X Kapa | 25 | 192.5 |
Total | 50 | 308 |
- 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]
Protocol | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Low-Input Nextera | X | X | X | X | X | X | X |
Protease Digestion (1:100 Dilution) | X | X | X | X | X | X | X |
Klenow Extension | X | X | X | X | X | X | X |
Heat | X | X | X | ||||
Heat w bisulfite | X | X | |||||
Desulphonation | X | X | X | ||||
Column | X | X | X | X | |||
Amplify | X | X | X | X | X | X | X |
- Nextera Low-Input, Normal Shendure 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
- NTC1 - normal shendure protocol
- NTC2 -shendure + bisulfite protocol
- Results show that heat and bisulfite conversion step (sulphonation or deamination?) cause the most lost during bisulfite conversion
- 6 NTC shoots up earlier than #5, which is weird. It was better than the last experiment where we saw NTC shoots up first. I say the extra sterilization steps cuts down the contamination but didn't completely eliminate the source.
- About 5 cycle difference when comparing library with and without bisulfite conversion, around 30 fold difference.
To Do[edit]
- I guess the epigentek Bisulflash DNA modification kit will be a good candidate to cut down the DNA lost during bisulfite conversion, since the conversion time is cut down to 20 minutes