AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions
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==Prepare PCR Product from Jurkat gDNA== | ==Prepare PCR Product from Jurkat gDNA== | ||
* | {| {{table}} | ||
| ||ul | |||
|- | |||
| primer||1 | |||
|- | |||
| h2o||7 | |||
|- | |||
| template||2 | |||
|- | |||
| Econo 2x||10 | |||
|- | |||
| | |||
|} | |||
*Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 2 -> 72C 10min -> 15C hold | |||
*Qiaquick Purification | |||
*Nanodrop | |||
P7-6.4ng/ul | |||
P10-5.4ng/ul | |||
*Gel Quant | |||
[[File:ZhangLab_2 2010-06-07 14hr 34min.jpg]] | |||
===Protocol=== | |||
Obtain 100ng Jurkat DNA | |||
*100ng/(6.4ng/ul)=15.63ul | |||
*100ng/(5.4ng/ul)=18.52ul | |||
{| {{table}} | |||
| ||P7||P10 | |||
|- | |||
| Jurkat DNA PCR Product||15.63||18.52 | |||
|- | |||
| ddH2O||69.37||66.48 | |||
|- | |||
| End Repair Reaction Buffer (10x)||10||10 | |||
|- | |||
| Repair Enzyme Mix||5||5 | |||
|- | |||
| | |||
|} | |||
*Incubate 30 mins at 20C | |||
Purification using qiaquick column | |||
*Elute with 50ul buffer | |||
Perform A-tailing protocol (6/8/10) | |||
*Add in 6ul dA-tailing Reaction buffer | |||
*Add in 4ul klenow fragment | |||
*Incubate at 37C for 30mins | |||
Purification using qiaquick column | |||
*Elute with 50ul buffer | |||
Adapter ligation | |||
*Mix 1ul methylation adapter | |||
*Mix 5ul ligase | |||
*Mix 14ul ligase buffer | |||
*Incubate @20c for 30mins | |||
Purification using qiaquick column | |||
*Elute with 50ul buffer | |||
CT Conversion (6/3/10) | |||
*Take 46ul to perfrom CT conversion | |||
an additional 1m | |||
Bisulfite conversion of DNA | |||
*Add 46ul of sample to 130ul of CT conversion reagent solution in a pcr tube | |||
*Vortex the sample to mix | |||
*Pulse centrifuge | |||
*Perform | |||
*98C for 8m | |||
*64C for 3.5hr | |||
*4C hold | |||
*Add 600ul of M binding buffer into a column assembly | |||
*Load sample(s) to the column | |||
*Close the cap and mix by inverting the column several times | |||
*Centrifuge at >10,000g for 30sec | |||
*Discard the flow through | |||
*Add 100ul of M-Wash Buffer to the column | |||
*Centrifuge at full speed for 30sec | |||
*Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m | |||
*Centrifuge at full speed for 30sec | |||
*Add 100ul of M-Wash Buffer to the column | |||
*Centrifuge at full speed for 30sec | |||
*Place the column into a 1.5ml tube | |||
*Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements) | |||
*Centrifuge for 30sec to elute the DNA | |||
==Perform PCR to bisulfite converted DNA using Phusion/iProof 6/9/10== | |||
*Primer | |||
{| {{table}} | |||
|- | |||
| PCR_F||Sol. Amp. (Forward)||AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] | |||
|- | |||
| PCR_R||Sol. Amp. (Reward)||CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T||3'-Phosphorothioate bond||[2] | |||
|- | |||
|} | |||
{| {{table}} | |||
| ||1rxn||4.4rxn|||| | |||
|- | |||
| Phusion||50||220|||| | |||
|- | |||
| PCRF||0.5||2.2|||| | |||
|- | |||
| PCRR||0.5||2.2|||| | |||
|- | |||
| SYBG||0.8||3.52|||| | |||
|- | |||
| |||||||| | |||
|- | |||
| ||P7||CTC P7||P10||CTC P10 | |||
|- | |||
| DNA||4||50||4||50 | |||
|- | |||
| H2O||46||0||46||0 | |||
|- | |||
| Mix||50||50||50||50 | |||
|- | |||
| | |||
|} | |||
a.Set up the reaction system with Phusion High-Fidelity PCR master mix: | |||
2x Phusion master mix: 50ul | |||
100uM PCR_F 0.5ul | |||
100uM PCR_R 0.5ul | |||
50X SYBG I 0.8ul | |||
Captured DNA 50ul | |||
b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. | |||
Terminate the reaction when the amplification curves approach to the plateau. | |||
[[File:060910.JPG|800px]] | |||
[[File:ZhangLab_2 2010-06-09 11hr 23min.jpg]] | |||
==Conclusion== | |||
*Result indicates that library construction is successful using 100ng of input DNA, band ~308bp for P7 Primer and ~280bp for P10 primer (Expected size at + 33bpx2 to the pcr product size) | |||
*It also shows that after bisulfite conversion, libaries are lost during the process and could not be amplified | |||
*Indicating the we need to use carriers to protect libraries | |||
*Unspecific bands showing up ~130bp could be due to primer dimer issue | |||
*Is size selection needed? | |||
*Is it possible to use Kapa instead of Phusion? I haven't noticed non specific bands after amplification using KAPA | |||
==Improvements== | |||
*Save a small amount of purified product after each step and run a gel to see which step brings in the non specific bands | |||
*Add carrier to protect libraries during bisulfite conversion | |||
*Size Selection | |||
*Using new Master Mix |
Latest revision as of 18:32, 18 June 2010
Prepare PCR Product from Jurkat gDNA[edit]
ul | |
primer | 1 |
h2o | 7 |
template | 2 |
Econo 2x | 10 |
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 2 -> 72C 10min -> 15C hold
- Qiaquick Purification
- Nanodrop
P7-6.4ng/ul P10-5.4ng/ul
- Gel Quant
File:ZhangLab 2 2010-06-07 14hr 34min.jpg
Protocol[edit]
Obtain 100ng Jurkat DNA
- 100ng/(6.4ng/ul)=15.63ul
- 100ng/(5.4ng/ul)=18.52ul
P7 | P10 | |
Jurkat DNA PCR Product | 15.63 | 18.52 |
ddH2O | 69.37 | 66.48 |
End Repair Reaction Buffer (10x) | 10 | 10 |
Repair Enzyme Mix | 5 | 5 |
- Incubate 30 mins at 20C
Purification using qiaquick column
- Elute with 50ul buffer
Perform A-tailing protocol (6/8/10)
- Add in 6ul dA-tailing Reaction buffer
- Add in 4ul klenow fragment
- Incubate at 37C for 30mins
Purification using qiaquick column
- Elute with 50ul buffer
Adapter ligation
- Mix 1ul methylation adapter
- Mix 5ul ligase
- Mix 14ul ligase buffer
- Incubate @20c for 30mins
Purification using qiaquick column
- Elute with 50ul buffer
CT Conversion (6/3/10)
- Take 46ul to perfrom CT conversion
an additional 1m Bisulfite conversion of DNA
- Add 46ul of sample to 130ul of CT conversion reagent solution in a pcr tube
- Vortex the sample to mix
- Pulse centrifuge
- Perform
*98C for 8m *64C for 3.5hr *4C hold
- Add 600ul of M binding buffer into a column assembly
- Load sample(s) to the column
- Close the cap and mix by inverting the column several times
- Centrifuge at >10,000g for 30sec
- Discard the flow through
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
- Centrifuge at full speed for 30sec
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Place the column into a 1.5ml tube
- Add 50ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements)
- Centrifuge for 30sec to elute the DNA
Perform PCR to bisulfite converted DNA using Phusion/iProof 6/9/10[edit]
- Primer
PCR_F | Sol. Amp. (Forward) | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T | 3'-Phosphorothioate bond | [2] |
PCR_R | Sol. Amp. (Reward) | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T | 3'-Phosphorothioate bond | [2] |
1rxn | 4.4rxn | |||
Phusion | 50 | 220 | ||
PCRF | 0.5 | 2.2 | ||
PCRR | 0.5 | 2.2 | ||
SYBG | 0.8 | 3.52 | ||
P7 | CTC P7 | P10 | CTC P10 | |
DNA | 4 | 50 | 4 | 50 |
H2O | 46 | 0 | 46 | 0 |
Mix | 50 | 50 | 50 | 50 |
a.Set up the reaction system with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 50ul 100uM PCR_F 0.5ul 100uM PCR_R 0.5ul 50X SYBG I 0.8ul Captured DNA 50ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. Terminate the reaction when the amplification curves approach to the plateau.
File:060910.JPG File:ZhangLab 2 2010-06-09 11hr 23min.jpg
Conclusion[edit]
- Result indicates that library construction is successful using 100ng of input DNA, band ~308bp for P7 Primer and ~280bp for P10 primer (Expected size at + 33bpx2 to the pcr product size)
- It also shows that after bisulfite conversion, libaries are lost during the process and could not be amplified
- Indicating the we need to use carriers to protect libraries
- Unspecific bands showing up ~130bp could be due to primer dimer issue
- Is size selection needed?
- Is it possible to use Kapa instead of Phusion? I haven't noticed non specific bands after amplification using KAPA
Improvements[edit]
- Save a small amount of purified product after each step and run a gel to see which step brings in the non specific bands
- Add carrier to protect libraries during bisulfite conversion
- Size Selection
- Using new Master Mix