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