Library/NOTES/2010-10-22: Difference between revisions
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===Adapter ligation=== | ===Adapter ligation=== | ||
'''Prepare adapters''' | '''Prepare adapters''' | ||
Stoffel buffer 6ul | |||
PE_t_adapter 2ul | |||
PE_b_adapter 2ul | |||
------------------- | ------------------- | ||
Total | |||
94C 2min -> 0.2C/sec to 20C -> 4C hold | 94C 2min -> 0.2C/sec to 20C -> 4C hold |
Revision as of 06:32, 29 October 2010
Mouse gut SAGs Library Construction
(copied from Dinh's page and modified based on the actual experiment)
ID MG1 MG2 MG4 MG5 MG8 MG9 MG12 MG13 MG14 MG15 MG16 MG32
Sheared DNA (approx. size 300bp) amount 1ug in total volume 100ul except MG12, 5ug in 100ul
End Repair
(NEB) x 13rxn mix sheared DNA 85ul - 10x End Repair reaction buffer 10ul 130ul End-repair enzyme mix 5ul 65 ------------------------------------------ - aliquot 15ul of reaction mix into PCR tube Total 100ul - add 85ul of sheared DNA (except MG12 add 17ul, adjust volume to 100ul with EB) - incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB.
Quick blunting Kit - repeat blunting because MDA buffer and enzyme may affect End-repair efficiency in previous step. x 13rxn mix DNA 38ul - 10X blunting bufer. 5ul 65ul dNTP mix 5ul 65ul Blunting Enzyme 2ul 26 ------------------------------------------ - aliquot 12ul of reaction mix into PCR tube, - add 38ul of DNA - incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB.
dA-tailing
x 14rxn mix End repaired DNA 37ul - dA-tailing buffer, 10x 5ul 70ul Klenow Fragment 3ul 42ul ------------------------------------------- - aliquot 13ul of reaction mix into PCR tube Total 50ul - add 37ul of end repaired DNA
- incubate in thermomixer at 37C for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB. - quantify each reaction with Nanodrop.
Adapter ligation
Prepare adapters
Stoffel buffer 6ul PE_t_adapter 2ul PE_b_adapter 2ul ------------------- Total
94C 2min -> 0.2C/sec to 20C -> 4C hold
Adapters to ligated product ratio: 20:1 Est length of dA-tailed products: 300bp (after shearing) MW dA-tailed products = (300bp*607.4 D/bp +157.9 D) = 182.378kD = 182,378 g/mole For 500 ng dA-tailed product = 500ng / 182,378g/mole = 27.4E-4 nmole * 20:1 = 0.055 nmole adapters required. Adapters (ul) = 0.055nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.055nmoles/ (20E-3 nmoles/ul) = 2.74 ul -----> 2.74 ul of 20uM adapters per 500ng dA-tailed products.
SampleID DNA(ul) Adapters(ul) QuickLigase(ul) 2xQuickLigase Buffer(ul)
Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.
Amplification
1x Reagents 10.0ul adapter ligased DNA 2.0ul PCR_F(10uM) 2.0ul PCR_R.N2IndX(10uM) [X=1,2,3,4,...12] 0.4ul SYBR Green 50x 35.6ul H2O 50.0ul Phusion HF, 2xMM -------------------------------- 100 ul each x 3 per sample
Program
98C - 30s, (98C - 10s, 62C - 20s, 70C - 30s)x10/12, 72C - 2min.
Size selection
Select 414bp band. Or 400-425bp band.