Library/NOTES/2010-10-22
Jump to navigation
Jump to search
Note: sorry, I forgot to label the images, and I'll note the image descriptions below the pictures soon.
Mouse gut SAGs Library Construction
(copied template from Dinh's page and modified based on the actual experiment)
Sample 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. (NEW) 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
(NEB) 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 and PAGE quantification.
File:ZhangLab 2 2010-10-25 12hr 18min MG SAGs no1,2,4,5,8,9,12,13,14,15 dAtailed.jpg File:ZhangLab 2 2010-10-25 12hr 18min MG SAGs no16,32,ntc dAtailed.jpg
Sample ID Conc.(ng/ul)by Nanodrop Conc.(ng/ul)by PAGE quantification MG1 -0.1 1.66* MG2 2.6 8.05* MG4 3.2 9.77* MG5 2.3 5.04* MG8 0.8 0.99** MG9 0.6 3.18* MG12 0.2 - MG13 0.3 0.14** MG14 -0.5 0.27** MG15 -0.0 2.62* MG16 -0.2 0.73** MG32 0.3 1.35* NTC 0.5 -
-From DNA conc. by PAGE quantification, DNA samples were divided in two subgroups - Gr.1 (*) Conc.>1.0ng/ul: MG1, MG2, MG4, MG5, MG9, MG15 & MG32 - Gr.2 (**) Conc.<1.0ng/ul: MG8, MG13, MG14 & MG16 - Sample MG12 didn't show the product in the gel then it was repeated again with larger amount of sheared DNA, 65ul in end repaired step. *** After Nanodrop check, it was grouped into Gr.2
Adapter ligation
Adapter calculation
Since A-tailed DNAs were divided into two groups baded on conc. range adapter was prepared in two different conc. according to maximum amount of A-tailed DNA in each group.
Adapters to ligated product ratio: 20:1 Gr.1 Est length of dA-tailed products: 500bp (based on the sized showed in the gel) MW dA-tailed products = (500bp*607.4 D/bp +157.9 D) = 303.857kD = 303,857 g/mole For 342 ng dA-tailed product (MG4) = 342ng / 303,857 g/mole = 11.2E-4 nmole * 20:1 = 0.0225 nmole adapters required. Adapters (ul) = 0.0225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.0225nmoles/ (20E-3 nmoles/ul) = 1.1 ul -----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.
Gr.2 For 35 ng dA-tailed product (MG8) = 35ng / 303,857 g/mole = 1.1E-4 nmole * 20:1 = 0.00225 nmole adapters required. Adapters (ul) = 0.00225nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.00225nmoles/ (20E-3 nmoles/ul) = 0.11 ul -----> 1.1 ul of 20uM adapters for dA-tailed products of Gr.1.
Adapter preparation
Stoffel buffer 6ul PE_t_adapter 2ul PE_b_adapter 2ul -------------------------------------- Total 10ul
94C 2min -> 0.2C/sec to 20C -> 4C hold x8.2 mix for Gr.1 x5.2 mix for Gr.2 A-tailed DNA 35ul - - Adapters 1ul 8.2ul 5.2ul (Adapter was diluted 10x in Stoffel buffer) QuickLigase 2ul 16.4 0.4 2xQuickLigase Buffer 38ul 312 198 -------------------------------------------------------------------------------------- Total 76ul - aliquot 41ul into each tube & add 35ul of A-tailed DNA - Incubate at RT fo 15 min. Purify with 0.7x AMPure beads. Elute with 40ul EB.
Amplification
x28 mix adapter ligased DNA 15 ul - PCR_F(10uM) 2.0ul 56ul PCR_R.N2IndX(10uM) [X=1,2,3,4,...12] 2.0ul - SYBR Green 50x 0.4ul 11.2ul H2O 30.6ul 857ul Phusion HF, 2xMM 50.0ul 1400ul --------------------------------------------------------------------------- Total 100ul - aliquot 83ul of rxn mix to each tube then add 15ul of adapter ligated DNA x2 for each sample and 2ul of PCR_R.N2IndX [X=1,2,3,4....,...12]
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C
- 2ul of amplified products were randomly analyzed in PAGE, and the rest were purified by AMPure, eluted with 40ul EB. - 2ul of AMPure purified products were analyzed again to quantify DNA conc. in the region approx. 400-600 bp.
File:ZhangLab 2 2010-10-26 14hr 08min.jpg
File:ZhangLab 2 2010-10-26 15hr 41min.jpgFile:ZhangLab 2 2010-10-26 15hr 44min.jpg
IndX Sample ID Conc. ng/ul Vol. ul (50ng) Ind1 MG1 18.16 2.8 Ind2 MG2 15.82 3.2 Ind3 MG4 16.21 3.1 Ind4 MG5 20.41 2.5 Ind5 MG8 21.16 2.4 Ind6 MG9 14.40 3.5 Ind7 MG12 2.38 21.0 Ind8 MG13 16.64 3.0 Ind9 MG14 21.73 2.3 Ind10 MG15 10.49 4.8 Ind11 MG16 30.77 1.6 Ind12 MG32 12.64 4.0 - Combind 50ng of each sample
Size selection
- Select 450bp band or 400 - 600bp band. - Perform PAGE size selection with 6% TBE 2D gel by cutting the band in the range of 400 - 600bp (x4 tubes, dissolved with 11ul EB, total vol. 44ul ).
File:ZhangLab 2 2010-10-27 13hr 08min MG SAGs SeqLib SS PAGE.jpg
- PAGE analysis to check the size and conc. of DNA library.
- From the gel, the size of DNA range was too big (approx. 400 almost 800bp). The reason was I used 25 bp as the ladder, so I's hard to identify the band above 500bp (couldn't find 100bp ladder). -Dr. Zhang recommended Dinh to cut DNA in the range of 450 - 550 bp.
- Perform PAGE size selection again with 6% TBE 5-well gel, eluted with 12ul EB.
File:ZhangLab 2 2010-10-27 13hr 08min MG SAGs SeqLib SS PAGE-round2.jpg
- 1ul of DNA was analyzed in PAGE and quantify DNA conc. = 13.8ng/ul or nM File:ZhangLab 2 2010-10-28 22hr 59min MG SAGs-SSPAGE-final.jpg