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

File:RC-1 PCR.png

  • 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.

File:PAGE-quan-RC-1.png