Kun:LabNotes/GenomeSeq/2010-9-10: Difference between revisions
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m (→PCR) |
m (→PCR) |
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Line 26: | Line 26: | ||
Ep.G-3 Ind6 Ind6+12 | Ep.G-3 Ind6 Ind6+12 | ||
1A Ind1+7 | 1A Ind1+7 | ||
1F Ind2+ | 1F Ind2+10(?) | ||
1G Ind3+11 | 1G Ind3+11 | ||
98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min. | 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. | Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles. | ||
[[Image:2010-09-10-ligation-PCR.png|350px]] | |||
Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O | Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O | ||
Conc(ng/ul) 300bp 800bp | Conc(ng/ul) 300bp 800bp | ||
Line 42: | Line 43: | ||
1F 5 | 1F 5 | ||
1G 6 | 1G 6 | ||
Combine | |||
B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library | |||
B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library | |||
1A, 1F, 1G => GM20431 Chr. library | |||
Perform size selection, cut out two different bands from the Lancelet 800bp library. | |||
Estimate size: | |||
Lancelet-300bp: 250-350bp | |||
Lancelet-800bp: 900-1000bp | |||
Lancelet-600bp: 600-700bp | |||
[[Image:2010-09-10-Lancelet-300bp-lib-size-selection.png|400px]] | |||
[[Image:2010-09-10-Lancelet-800bp-lib-size-selection.png|400px]] | |||
[[Image:2010-09-10-GM20431-chr-lib-size-selection.png|400px]] |
Revision as of 00:56, 11 September 2010
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+10(?) 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. File:2010-09-10-ligation-PCR.png
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 Combine B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library 1A, 1F, 1G => GM20431 Chr. library
Perform size selection, cut out two different bands from the Lancelet 800bp library. Estimate size: Lancelet-300bp: 250-350bp Lancelet-800bp: 900-1000bp Lancelet-600bp: 600-700bp File:2010-09-10-Lancelet-300bp-lib-size-selection.png File:2010-09-10-Lancelet-800bp-lib-size-selection.png File:2010-09-10-GM20431-chr-lib-size-selection.png