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== Amplification with adapter primers == * Reaction #1: CP-2-FA.IndSol (Indx =7, 45, and 76) & CP-2-RA.Sol * Reaction #2: Switch.CP-2-FA.Sol & Switch.CP-2-RA.IndSol (Indx = 7, 45, and 76) * I did the reaction #1 first to see if the amplification work. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''7.5 rxn mix''' |- | template||4.00||0.00 |- | 10uM CA-2-FA.Indx Sol||2.00||0.00 |- | 10uM CA-2-RA.Sol||2.00||15.00 |- | 2x KAPA MM||50.00||375.00 |- | H2O||42.00||315.00 |- | Total||100.00||750.00 |} * Aliquot 94ul, add 4 ul template and 2ul Indx primer '''Program'''<br> 98C 30sec --> (98 C 10 sec --> 54C 30sec --> 72C 30sec) x 4 --> 72 C 3 min<br> * For both FA.IndSol or RA.IndSol, each sample used the same index number. [[File:reaction1.png| 500px]]<br> * Reaction #2 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''7.5 rxn mix''' |- | template||4.00||0.00 |- | 10uM Switch.CA-2-FA.Sol||2.00||15.00 |- | 10uM Switch.CA-2-RA.IndSol||2.00||0.00 |- | 2x KAPA MM||50.00||375.00 |- | H2O||42.00||315.00 |- | Total||100.00||750.00 |} Aliquot 94ul, add 4 ul template and 2ul Indx primer * Used the same PCR cycler as reaction #1 [[File:reaction2.png| 500px]]<br> * Purified PCR product with Ampure bead and eluted with 40ul EB buffer * Perform PAGE quantification by loading 1ul of purified amplicon in 6% TBE gel. I also loaded the size-selected DNA used as the template to see if I cut the right band. [[File:ZhangLab_2 2011-10-11 05hr 37min-PQ-1.jpg| 400px]][[File:ZhangLab_2 2011-10-11 05hr 40min-PQ-2.jpg| 400px]]<br> * 1 = PGP1 F 52C, 2 = PGP1F 54C, 3 = PGP1iPS 52C, 4 = PGP1iPS 54C, 5 = H1 52C, 6 = H1 54C<br> * A = PGP1F, B = PGP1iPS, C = H1<bR> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample /Reaction#1''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''yields in 40ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25 ng''' |- | PGP1F 52C ||1.56||62.55||15.99 |- | PGP1F 54C||0.90||35.88||27.87 |- | PGP1iPS 52C ||1.61||64.40||15.53 |- | PGP1iPS 54C||1.08||43.33||23.08 |- | H1 52C||3.64||145.51||6.87 |- | H1 54C||2.74||109.69||9.12 |- | |- | Sample /Reaction#2||conc. (ng/ul)||yields in 40ul (ng)||Volume for 25 ng |- | PGP1F 52C swi||0.82||32.62||30.65 |- | PGP1F 54C swi||0.61||24.36||41.06 |- | PGP1iPS 52C swi||0.64||25.76||38.81 |- | PGP1iPS 54C swi||0.97||38.81||25.76 |- | H1 52C swi||3.09||123.57||8.09 |- | H1 54C swi||2.15||86.02||11.63 |} * I pooled the samples with the same annealing temperature during capture together, so I would have the libraries from 52 and 54C * Did PAGE size-selection even I could see the clear band ~350bp since I still see a little smear below the expected band. [[File:ZhangLab_2 2011-10-11 07hr 27min-PAGE-SeqLib.jpg| 400px]]<br> * Resuspended DNA with H2O --> total 20ul, and quantified conc. by Qubit dsDNA HS * SampleID: NP-BSPP-LC-52C_Oct6, 2011: 2.2ng/ul --> yields = 20ul x 2.2ng/ul = 44ng, --> 10.3nM (calculated based on size 350bp) * SampleID: NP-BSPP-LC-54C_Oct6, 2011: 1.73ng/ul --> yields = 20ul x 1.73ng/ul = 34.6ng --> 8.1nM (calculated based on size 350bp) [[File:ZhangLab_2 2011-10-11 11hr 06min-SeqLib-PC.jpg| 300px]]
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