Noi/NOTES/2012-5-14

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Revision as of 15:17, 14 May 2012 by >Noi (Created page with "* http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''' == Probe production for the HiResChrPaint project == * Continued from the plan...")
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Probe production for the HiResChrPaint project

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.