Kun:LabNotes/ASE/2007-10-29: Difference between revisions
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[[image:2007-10-29-PCR-Lambda_exo-TBE.jpg]] [[image:2007-10-29-PCR-Lambda_exo-TBU.jpg]] | [[image:2007-10-29-PCR-Lambda_exo-TBE.jpg]] [[image: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. | *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 probably used the wrong reverse primer for the eSNP84k sets. I should have used the phosAP2V4 instead of AP2V4, since lambda exonuclease doesn't cut without a 5'-phosphate group. | ||
===Exp2: Repeat PCR amplification on the eSNP84k library=== | ===Exp2: Repeat PCR amplification on the eSNP84k library=== | ||
Reactions eSNP84k set1-5 (100nM): | Reactions eSNP84k set1-5 (100nM): |
Revision as of 01:12, 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. I probably used the wrong reverse primer for the eSNP84k sets. I should have used the phosAP2V4 instead of AP2V4, since lambda exonuclease doesn't cut without a 5'-phosphate group.
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