Noi/NOTES/2013-3-14

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Revision as of 20:11, 15 March 2013 by >Noi
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Set 1

Components 1rxn 6.5 rxn
Captured template 12.00 0.00
10uM Forward no barcodes 2.00 13.00
10uM Reverse +Indx 2.00 0.00
2x KAPA SYBG fast MM 50.00 325.00
H2O 34.00 221.00
Total 100.00 650.00
  • Aliquot 86ul, add 2ul of reverse Ind, 12ul of circular DNA

Set 2

Components ' '
Captured template 1rxn 6.5 rxn
10uM Forward + Indx 12.00 0.00
100uM Reverse no barcodes 2.00 0.00
2x KAPA SYBG fast MM 2.00 13.00
H2O 50.00 325.00
Total 34.00 221.00
100.00 650.00
  • Aliquot 86ul, add 2ul of Forward Ind, 12ul of circular DNA
  • Program

98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min (total 21 cycles)

  • Perform bead purification , set 1 elute total volume 60ul, set 2 elute total volume 50ul.
  • Verify by PAGE an do PAGE quantification to see the ratio of each product before pooling the libraries.
  • Note: I quantified based on the expected band ~250bp and ~400 (two copy of target based on amplification of circular DNA.

File:ZhangLab 2 2013-03-15 11hr 27min SeqLib Insitu.jpg

Samples Conc. (ng/ul) Yields(ng) Pooled volume(ul)
gDNA-set1 26.50 1590.05 37.73
dsDNA -RNaseA set1 34.14 2048.43 29.28
dsDNA +RNaseA set1 33.33 1999.55 30.00
gDNA-set2 27.11 1355.44 10.41
dsDNA -RNaseA set2 7.05 352.73 40.00
dsDNA +RNaseA set2 10.38 519.25 27.17