AlanFung:LabNotes/CTCF/2010-6-7: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 129: | Line 129: | ||
Terminate the reaction when the amplification curves approach to the plateau. | Terminate the reaction when the amplification curves approach to the plateau. | ||
[[File:060910.JPG|800px]] | [[File:060910.JPG|800px]] | ||
[[File:ZhangLab_2 2010-06-09 11hr 23min.jpg | [[File:ZhangLab_2 2010-06-09 11hr 23min.jpg]] | ||
==Conclusion== | ==Conclusion== |
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