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2nd MDA amplification
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=2nd MDA amplification for Test2 and Test3 results= ==Objective== *To increase the MDA product for downsteam analysis ==Samples & Materials== *Amplicons from Test2 (Cell line ): B7, C8 *Amplicons from Test3 (Cell lone ): B1, B4 ==Procedure== * Set up PCR program (on traditional thermocycler) * Thaw the N6 primer, reagents from phi29 kit except enzyme * Prepare master mix 1 rxn 4.5 rxn ----------------------------------- H2O 147.0 661.5 10X buffer 20.0 90.0 N6 Primer 10.0 45.0 dNTP 8.0 36.0 Phi29 Enzyme 10.0 45.0 ------------------------------------ 195.0 877.5(uL) 877.5/4.5=195 *Set up 4 PCR tube on the cool-block, labeled *Transfer 5 uL MDA amplicon (test2-B7, test2-C8, test3-B1, test3-b4) to each reaction tubes. *Transfer 195 uL Master mix in tubes. Mix well by repeat pipetting. *Perform amplification on thermocycler 30C 2hr -> 85C 3 min -> 4C forever ==PCR confirmation== *Primer: 18S, Primer 2-1 *Template: 1/10 dilution of 2nd MDA amplicons (B7, C8, B1, B4), Pos (1 ng/uL gDNA), H2O *PCR Master mix x 2 (Primer various) 1 rxn 7 rxns H2O 3.5 24.5 Primer 0.5 3.5 Taq2X 5.0 35.0 -------------------- 9.0 63.0 uL 63/7=9 -> Template 1 uL *Set up PCR reaction on the cool-block ** Since only one Thermocycler block is available (others are occupied, we use the 56C Tm for both two reaction which acturally need 52C and 59C for Tm) ==Gel electrophoresis== *1.5% agarosel (100 mL + 8 uL SYBR safe), 135V, run 20 min. *Loading order: T2B7, T2C8, T3B1, T3B4, Pos, H2O (Left to Right) ==Results== [[Image:ZhangLab_2 2009-03-01 10hr 33min-p18-p2-1.bmp|500px]] *Left: Test by 18S primer (306 bp). Right: Test by 2-1 primer (540 bp) *Some bands are very weak probably due to the inappropriate Tm we used. *We will give T2B7 and T3B1 samples to Illumina for analysis.
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