Alice:LabNotes/2010-4-23: Difference between revisions
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=PCR re-amplification of the sequencing libraries with PAIR-END primer= | =PCR re-amplification of the sequencing libraries with PAIR-END primer= | ||
*samples to be re-amplified: CViB | *samples to be re-amplified: | ||
**CViB: 5.27nM =10.43ng/ul | |||
**DF: 81.7nM = 161.77ng/ul | |||
DNA | **foreskin: 75.38nM = 149.25ng/ul | ||
**ips17A: 2.04nM = 4.04ng/ul | |||
**ips17B: 2.01nM = 3.9ng/ul | |||
**ips11B: 3.81nM = 7.54ng/ul | |||
**Hfib 11: 3.22nM = 6.38ng/ul | |||
**CV1: 1.34nM= 3.1ng/ul (done on 4/26/2010) | |||
x4 well each | |||
DNA 1ul | |||
Phusion HF PCR Master Mix (2x): 50ul | Phusion HF PCR Master Mix (2x): 50ul | ||
PCR_R (100uM): | PCR_R (100uM): 0.2ul | ||
PCR_F (100uM): | PCR_F (100uM): 0.2ul | ||
SYBR Green 50x: | SYBR Green 50x: 0.4ul | ||
H2O: | H2O: 48ul | ||
Total: | 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 | ||
*Qiaquick column purification and elute in 50ul EB. | |||
*nanodrop result: | |||
*CViB: 32.4ng/ul | |||
*DF: 29.5ng/ul | |||
*Hfib 11: 19.1ng/ul | |||
*11b: 24.2ng/ul | |||
*17b: 15.5ng/ul | |||
*17a: 17.6ng/ul | |||
*FS: 33.9ng/ul | |||
*CV1: 23.1ng/ul (done on 4/26/2010) | |||
*AMPure beads purification using 1x total reaction volume (50ul beads) and elute in 40ul ddH2O | |||
==result== | |||
[[File:ZhangLab_2 2010-04-25 16hr 20min.jpg]] | |||
==PAGE quantification== | |||
[[File:ZhangLab_2 2010-04-27 13hr 37min.jpg]] | |||
*37.16nM= hFib11 (350bp) | |||
*57.73nM= ips11b (350bp) | |||
*61.86nM= CviB (350bp) | |||
*26.90nM= ips17b (350bp) | |||
*74.71nM= FS (300bp) | |||
*32.37nM= ips17a (350bp) | |||
*67.09nM= DF (300bp) | |||
*51.07nM= CV1 (350bp) | |||
*84.67nM= CviF (350bp) | |||
=Sanger validation of candidate mutations= | =Sanger validation of candidate mutations= | ||
==DNA preparation== | ==DNA preparation== | ||
* prepared the following stock solutions for PCR templates | * prepared the following stock solutions for PCR templates | ||
Sample Conc. | Sample Conc. Volume H2O Final Conc. | ||
PGP1F 41.8ng/ul 2ul | PGP1F 41.8ng/ul 2ul 39.8ul 2ng/ul | ||
PGP1-ips 399ng/ul | PGP1-ips 399ng/ul 1ul 198.5ul 2ng/ul | ||
==PCR== | ==PCR== | ||
Line 40: | Line 73: | ||
*Add 1ul of purified PCR product with 9ul of ddH2O. | *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 | *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 | ||
==result== | |||
[[File:mutations.jpg | 700px]] |
Latest revision as of 18:36, 28 April 2010
PCR re-amplification of the sequencing libraries with PAIR-END primer[edit]
- samples to be re-amplified:
- CViB: 5.27nM =10.43ng/ul
- DF: 81.7nM = 161.77ng/ul
- foreskin: 75.38nM = 149.25ng/ul
- ips17A: 2.04nM = 4.04ng/ul
- ips17B: 2.01nM = 3.9ng/ul
- ips11B: 3.81nM = 7.54ng/ul
- Hfib 11: 3.22nM = 6.38ng/ul
- CV1: 1.34nM= 3.1ng/ul (done on 4/26/2010)
x4 well each DNA 1ul 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
- Qiaquick column purification and elute in 50ul EB.
- nanodrop result:
- CViB: 32.4ng/ul
- DF: 29.5ng/ul
- Hfib 11: 19.1ng/ul
- 11b: 24.2ng/ul
- 17b: 15.5ng/ul
- 17a: 17.6ng/ul
- FS: 33.9ng/ul
- CV1: 23.1ng/ul (done on 4/26/2010)
- AMPure beads purification using 1x total reaction volume (50ul beads) and elute in 40ul ddH2O
result[edit]
File:ZhangLab 2 2010-04-25 16hr 20min.jpg
PAGE quantification[edit]
File:ZhangLab 2 2010-04-27 13hr 37min.jpg
- 37.16nM= hFib11 (350bp)
- 57.73nM= ips11b (350bp)
- 61.86nM= CviB (350bp)
- 26.90nM= ips17b (350bp)
- 74.71nM= FS (300bp)
- 32.37nM= ips17a (350bp)
- 67.09nM= DF (300bp)
- 51.07nM= CV1 (350bp)
- 84.67nM= CviF (350bp)
Sanger validation of candidate mutations[edit]
DNA preparation[edit]
- prepared the following stock solutions for PCR templates
Sample Conc. Volume H2O Final Conc. PGP1F 41.8ng/ul 2ul 39.8ul 2ng/ul PGP1-ips 399ng/ul 1ul 198.5ul 2ng/ul
PCR[edit]
- 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