Kun:LabNotes/GenomeSeq/2010-9-10
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Library construction (cont.)
- The libraries made on 9/9 were good. I need to repeat the ligation using the ligation products made on 8/26 for BJ3.3 and BJ3.8. Then I will do ligation PCR with the barcoded primers on all the ligation products.
Ligation
Purified ligation products from 8/36 18 μl Quick Ligation Reaction Buffer (5X) 6 μl 20 μM DNA Adaptors x μl Quick T4 DNA Ligase 2 μl H2O 4-x ul Incubate RT for 15 minutes. Purified with 1V AMPure beads.
PCR
Adapter ligated DNA 20 μl PCR_F(10uM) 2 μl PCR_R.N2IndX(10uM) 2 μl 50X SYBR Green I 0.8 μl H2O 25 μl 2X Phusion HF MM 50 μl ----- 100 μl Barcodes 300bp 800bp B.Jap-1 Ind1 Ind1+7 B.Jap-2 Ind2 Ind2+8 B.Jap-3 Ind3 Ind3+9 Ep.G-1 Ind4 Ind4+10 Ep.G-2 Ind5 Ep.G-3 Ind6 Ind6+12 1A Ind1+7 1F Ind2+9 1G Ind3+11 98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min. Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles. Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O Conc(ng/ul) 300bp 800bp B.Jap-1 12 6 B.Jap-2 12 9 B.Jap-3 3 5 Ep.G-1 10 8 Ep.G-2 4 Ep.G-3 7 7 1A 8 1F 5 1G 6