Noi/NOTES/2012-5-14
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Probe production for the HiResChrPaint project
- Continued from the plan: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-8
Production PCR
- V4 pool for all four sets (12 reaction for each set)
Components | Volume (ul) | 49x rxn mix |
20nM oligo pool | 0.20 | 0.00 |
2x Kapa SYBG qPCR MM | 50.00 | 2,450.00 |
100uM pAP1V41U | 0.40 | 19.60 |
100uM AP2V4 | 0.40 | 19.60 |
2mM aa-dUTP | 3.00 | 147.00 |
H2O | 46.00 | 2,254.00 |
Total volume (ul) | 100.00 | 4,900.00 |
- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube
Program
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
- V6 pool for all four sets (12 reaction for each set)
Components | Volume (ul) | 49x rxn mix |
20nM oligo pool | 0.20 | 0.00 |
2x Kapa SYBG qPCR MM | 50.00 | 2,450.00 |
100uM pAP1V61U | 0.40 | 19.60 |
100uM AP2V6 | 0.40 | 19.60 |
2mM aa-dUTP | 3.00 | 147.00 |
H2O | 46.00 | 2,254.00 |
Total volume (ul) | 100.00 | 4,900.00 |
- Aliquot 1197.6 ul in 1.5 mL tube, add 2.4ul of each oligo pool to each tube
Program
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
- Check the size distribution of the amplicons with 6% TBE gels. - Ethanol precipitation. - Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB. - Measure concentration.