Sam:LabNotes/Human/2009-5-10 exp1
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Perform 2nd round MDA on the amplicons from 200pg L4N9 [edit]
Objective[edit]
- Amplify the MDA amplicons from 200pg-L4N9, L4N8, L4N7 for Illumina Infinium array genotyping
- The request DNA concentration for Infinium assay: 5 ug/uL for 15 uL
- In order to optimize the condition of DNA purification, only L4N9 was tested with different reaction time (4h vs 8h)
Procedure[edit]
- Set up PCR program on traditional thermocycler
- MDA4h 30C 4hr -> 85C 3 min -> 4C forever
- MDA8h 30C 8hr -> 85C 3 min -> 4C forever
- Thaw the L4N9 primer, reagents from phi29 kit except enzyme
- Set up reaction in PCR tubes
4hr-rxn/8hr-rxn ----------------------- H2O 147.0 10X buffer 20.0 L4N9 Primer 10.0 dNTP 8.0 Phi29 Enzyme 10.0 ----------------------- 195.0
- Transfer 5 uL MDA amplicon, amplicons from 200pg-L4N9, into each of two reactions. Mix well by repeat pipetting.
- Perform amplification on thermocycler based on assigned program (MDA4h/MDA8h)