Kun:LabNotes/Haplotyping/2009-1-29
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Perform 2nd round MDA on the amplicons from 01/28/2009
- The purpose of this experiment is to generate more DNA for downstream analyses. Only the 16 amplicons from the haploid templates will be re-amplified.
Set up master mix: x 16 10x RepliPhi phi-29 buffer 20ul 320 1uM N6 primer 10ul 160 25mM dNTP 8ul 128 2X SYBR Green I 10ul 160 H2O 142ul 2272 RepliPhi Phi-29 (100U/ul) 10ul 30 Add 200ul MDA mix to each of the tube on cool block, 30C 2h, 85C 3min.
Make aliquots of amplicons (50ul each) for Infinium Genotyping: GM18508A GM18507B GM18507D GM18507G GM20431A GM20431B GM20431C GM20431F Ethanol precipitation of 150ul amplicons: GM18508A GM18507B GM18507D GM18507G GM20431A GM20431B GM20431C GM20431G
S1 nuclease debranching
- Resuspend the precipitated DNA in 100ul ddH2O.
Set up S1 nuclease digestion MDA amplicon: 20ul 10X S1 nuclease buffer: 5ul 3M NaCl: 5ul H2O: 15ul S1 nuclease (Invitrogen, 10U/ul): 5ul 37c 1h -> 2C -> Purified with Qiaquick columns. Check the size of debranched DNA with 1% argarose gel.
The DNA are smears of 10kb or less. The gel image was accidentally erased.
DNA shearing
- I sheared three samples (Cell-a, Cell-b, gDNA-a) with nebulizers at 35psi for 6 min.
- The sheared DNA (~600ul) were precipitated with 1V isopropanol.
- The recovery was pretty poor. Also the sheared DNA fragments are still too big (500bp-1.2kb).
- I decided to send all the amplicons (5 S1 treated, and 11 untreated) to Covaris for shearing. The target size is 300bp.