Noi/NOTES/2011-12-13
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Adding barcode to RC-1 sequencing library from Baylor[edit]
- Received one sequencing library form Baylor, RC-1 --> labeled conc. 70 ng/ul, volume ~ 8ul. I added 8ul of H2O to increase the volume, so the final conc. ~35ng/ul.
- This library was constructed using the same protocol as I did on the LC sciences's BSPP library. I will perform qPCR with the barcode primers.
- Reaction #1: CA-2-FA.Indx7.Sol & CA-2-RA.Sol
- Reaction #2: Switch.CA-2-FA.Sol & Switch.CA-2-RA.Ind7.Sol
Components Reaction #1 | 1 rxn | 2x rxn mix | Components Reaction #2 | 1 rxn | 2x rxn mix |
Template | 2.00 | 0.00 | Template | 2.00 | 0.00 |
10 uM CA-2-FA.Indx7.Sol | 2.00 | 4.00 | 10 uM Switch.CA-2-FA.Sol | 2.00 | 4.00 |
10 uM CA-2-RA.Sol | 2.00 | 4.00 | 10 uM Switch.CA-2-RA.Ind7.Sol | 2.00 | 4.00 |
2X KAPA MM | 50.00 | 100.00 | 2X KAPA MM | 50.00 | 100.00 |
H2O | 44.00 | 88.00 | H2O | 44.00 | 88.00 |
Total | 100.00 | 200.00 | Total | 100.00 | 200.00 |
- aliquot 98ul, add 2ul of DNA template
Program
98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min
File:QPCR curve Rc-1.png
- Purified PCR product with Ampure bead and eluted with 40ul EB buffer
- Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel
- The concentrations of amplified amplicons reaction #1 and #2 are 1.26ng/ul (yield = 50.5ng) and 1.07ng/ul (yield = 42.8), respectively.
- I then pooled 50ng of each reaction and performed PAGE size selection by cutting the size ~375bp (below the 400bp ladder band).
- Alan helped me quantified the concentration by Qubit = 5.45nM and also verified by running in PAGE.