Alice:LabNotes/2010-4-23: Difference between revisions
Jump to navigation
Jump to search
>Zsakura2 (Created page with '=PCR re-amplification of the sequencing libraries with PAIR-END primer= *samples to be re-amplified: CViB, DF, foreskin, ips17A, ips17B, ips11B, Hfib 11 …') |
>Zsakura2 |
||
Line 11: | Line 11: | ||
Total: 100ul | Total: 100ul | ||
98C 30sec -> (98C 10sec -> 55C 45sec -> 72C 30sec) x2 cycles -> (98C 10 sec -> 72C 30sec) x 3cycles -> 72C 5min -> hold at 4C | 98C 30sec -> (98C 10sec -> 55C 45sec -> 72C 30sec) x2 cycles -> (98C 10 sec -> 72C 30sec) x 3cycles -> 72C 5min -> hold at 4C | ||
=Sanger validation of candidate mutations= | |||
==DNA preparation== | |||
* prepared the following stock solutions for PCR templates | |||
Sample Conc. Volume H2O Final Conc. | |||
PGP1F | |||
PGP1-ips | |||
==PCR== | |||
* 6 reactions total. | |||
x3 x2 | |||
DNA(2ng/ul) 3ul 15ul | |||
Taq 2x master mix 25ul 125ul | |||
10uM F primer 1ul - | |||
10uM R primer 1ul - | |||
H2O 20ul 100ul | |||
total 50ul 250ul | |||
94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min. | |||
*use 2ul to run a TBE gel to check the PCR results, then save 18ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB. | |||
*Use Nanodrop to check the concentration of the purified PCR product. | |||
*TBE gel result: | |||
*Prepare the samples for sequencing: | |||
*USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers. | |||
*The DNA size is 200-500bp, so we use 10ng of DNA in 10ul volume (~1ng/ul) | |||
*Add 1ul of purified PCR product with 9ul of ddH2O. | |||
*Add the Forward primer for sequencing, dilute the Forward primer to 5uM, add 5ul of primer(total of 25pmol) and get a final volume of 15 |
Revision as of 17:44, 23 April 2010
PCR re-amplification of the sequencing libraries with PAIR-END primer
- samples to be re-amplified: CViB, DF, foreskin, ips17A, ips17B, ips11B, Hfib 11
x4 well each DNA (0.353ng/ul) 2.5ul Phusion HF PCR Master Mix (2x): 50ul PCR_R (100uM): 0.2ul PCR_F (100uM): 0.2ul SYBR Green 50x: 0.4ul H2O: 48ul Total: 100ul 98C 30sec -> (98C 10sec -> 55C 45sec -> 72C 30sec) x2 cycles -> (98C 10 sec -> 72C 30sec) x 3cycles -> 72C 5min -> hold at 4C
Sanger validation of candidate mutations
DNA preparation
- prepared the following stock solutions for PCR templates
Sample Conc. Volume H2O Final Conc. PGP1F PGP1-ips
PCR
- 6 reactions total.
x3 x2 DNA(2ng/ul) 3ul 15ul Taq 2x master mix 25ul 125ul 10uM F primer 1ul - 10uM R primer 1ul - H2O 20ul 100ul total 50ul 250ul 94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min.
- use 2ul to run a TBE gel to check the PCR results, then save 18ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB.
- Use Nanodrop to check the concentration of the purified PCR product.
- TBE gel result:
- Prepare the samples for sequencing:
- USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers.
- The DNA size is 200-500bp, so we use 10ng of DNA in 10ul volume (~1ng/ul)
- Add 1ul of purified PCR product with 9ul of ddH2O.
- Add the Forward primer for sequencing, dilute the Forward primer to 5uM, add 5ul of primer(total of 25pmol) and get a final volume of 15