2nd MDA amplification
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2nd MDA amplification for Test2 and Test3 results[edit]
Objective[edit]
- To increase the MDA product for downsteam analysis
Samples & Materials[edit]
- Amplicons from Test2 (Cell line ): B7, C8
- Amplicons from Test3 (Cell lone ): B1, B4
Procedure[edit]
- 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[edit]
- 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[edit]
- 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[edit]
File:ZhangLab 2 2009-03-01 10hr 33min-p18-p2-1.bmp
- 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.