Kun:LabNotes/ASE/2007-10-29: Difference between revisions
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*I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours. | *I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours. | ||
===Exp2: Repeat PCR amplification on the eSNP84k library=== | |||
Reactions eSNP84k set1-5 (100nM): | |||
x 8 x 5 | |||
Template 0.2ul 1.6ul x 5 | |||
10X buffer 10ul 80ul x 5 | |||
10mM dNTP 2ul 16ul x 5 | |||
100uM AP1V41U 0.4ul 3.2ul x 5 | |||
100uM phosAp2V4 0.4ul 3.2ul x 5 | |||
AmpliTaq Gold 1ul 16ul x 5 | |||
H2O 86ul 680ul x 5 | |||
50X SYBR I 0.8ul 6.4ul x 5 | |||
95C 15min -> 17 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold |
Revision as of 01:10, 30 October 2007
Exp1: Probe prep (cont.)
- Starting from ethanol precipitated DNAs made at 10/18/2007.
- Pool two tubes of the same amplicons into ~100ul in total, add 11ul 10x Lambda Exo buffer, 4ul 5U/ul Lambda exonuclease. Incubate @ 37C for 2 hours, check 2ul of the products in both 8% TBE gels and 6% TBU gels.
8% TBE gel 6% TBU gel File:2007-10-29-PCR-Lambda exo-TBE.jpg File:2007-10-29-PCR-Lambda exo-TBU.jpg
- I saw sharp bands in the non-denaturing gel, suggesting there are still a lot of dsDNA. I decided to incubate the reactions for another 4 hours.
Exp2: Repeat PCR amplification on the eSNP84k library
Reactions eSNP84k set1-5 (100nM):
x 8 x 5 Template 0.2ul 1.6ul x 5 10X buffer 10ul 80ul x 5 10mM dNTP 2ul 16ul x 5 100uM AP1V41U 0.4ul 3.2ul x 5 100uM phosAp2V4 0.4ul 3.2ul x 5 AmpliTaq Gold 1ul 16ul x 5 H2O 86ul 680ul x 5 50X SYBR I 0.8ul 6.4ul x 5
95C 15min -> 17 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 4C hold