Noi/NOTES/2013-3-14
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- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-12
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 |