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==Amplification== '''x28 mix''' adapter ligated 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|350px|Image: 350 pixels]]<br> <br> Figure3. 25bp ladder, MG1, MG5, MG9, MG13, MG14, MG12, MG15, MG32, NTC [[File:ZhangLab_2 2010-10-26 15hr 41min.jpg|450px|Image: 450 pixels]][[File:ZhangLab_2 2010-10-26 15hr 44min.jpg|300px|Image: 300 pixels]]<br> Figure4. LM 6ul, LM 3ul, MG1, MG2 MG4, MG5, MG8, MG9, MG12, MG13 Figure5. LM6ul, LM 3ul, MG14, MG15, MG16, MG32 '''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|300px|Image: 300 pixels]]<br> Figure6, MG SAGs BatchA Sequencing Library PAGE size selection (25bp ladder) - 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|300px|Image: 300 pixels]]<BR> Figure7. MG SAGs BatchA Sequencing Library PAGE size selection (round2) 25bp ladder, 1kb ladder, 1st round PAGE SS purified sequencing library, 50bp ladder - 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|250px|Image: 250 pixels]]<br> Figure8. LM 6ul, LM3ul, 2 round PAGE SS purified sequencing library, 1kb ladder, 25bp ladder
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