Kun:LabNotes/Haplotyping/2009-1-29
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Perform 2nd round MDA on the amplicons from 01/28/2009[edit]
- 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[edit]
- 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[edit]
- 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.
GM18507A Purified DNA (50ul) + 50ul 1X TE GM18507B Purified DNA (50ul) + 50ul 1X TE GM18507C Unpurified DNA (50ul) + 50ul 1X TE GM18507D S1 treated DNA (40ul) + 50ul 1X TE GM18507E Unpurified DNA (50ul) + 50ul 1X TE GM18507F Unpurified DNA (50ul) + 50ul 1X TE GM18507G S1 treated DNA (40ul) + 50ul 1X TE GM18507H Unpurified DNA (50ul) + 50ul 1X TE GM20431A Purified DNA (50ul) + 50ul 1X TE GM20431B S1 treated DNA (40ul) + 50ul 1X TE GM20431C Unpurified DNA (50ul) + 50ul 1X TE GM20431D Unpurified DNA (50ul) + 50ul 1X TE GM20431E Unpurified DNA (50ul) + 50ul 1X TE GM20431F Unpurified DNA (50ul) + 50ul 1X TE GM20431G S1 treated DNA (40ul) + 50ul 1X TE GM20431H Unpurified DNA (50ul) + 50ul 1X TE
Illumina Infinium genotyping results[edit]
Alleles on heterozygous SNPs[edit]
Alleles | GM18507A | GM18507B | GM18507D | GM18507G |
0 | 131607 | 128075 | 99430 | 56922 |
1 | 22482 | 11383 | 13441 | 2874 |
2 | 11551 | 26182 | 52769 | 105844 |
Alleles | GM20431A | GM20431B | GM20431C | GM20431F |
0 | 139685 | 110445 | 49761 | 18786 |
1 | 22272 | 22277 | 3004 | 815 |
2 | 20738 | 49973 | 129930 | 163094 |