Noi/NOTES/2013-3-14
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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)x15 -> 72C 3min
- 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 ~400bp. However the intensity of the ~250bp band was very strong I also quantified and compared the intensities to the 400bp band.
File:ZhangLab 2 2013-03-15 11hr 27min SeqLib Insitu.jpg
Samples | Conc. (ng/ul) | Yields (ng) | Pooled volume (ul) | 250bp/400bp (folds) |
gDNA-set1 400bp | 7.34 | 440.57 | 40.56 | 2.51 |
dsDNA -RNaseA set1 400bp | 9.93 | 595.71 | 30.00 | 2.37 |
dsDNA +RNaseA set1 400bp | 9.99 | 599.24 | 30.00 | 2.27 |
gDNA-set2 400bp | 6.11 | 305.61 | 3.45 | 3.32 |
dsDNA -RNaseA set2 400bp | 0.53 | 26.39 | 40.00 | 11.03 |
dsDNA +RNaseA set2 400bp | 1.24 | 61.96 | 17.04 | 6.81 |
gDNA-set1 250bp | 18.45 | |||
dsDNA -RNaseA set1 250bp | 23.51 | |||
dsDNA +RNaseA set1 250bp | 22.63 | |||
gDNA-set2 250bp | 20.29 | |||
dsDNA -RNaseA set2 250bp | 5.82 | |||
dsDNA +RNaseA set2 250bp | 8.44 |