Kun:LabNotes/Haplotyping/2009-1-29: Difference between revisions
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*Resuspend the precipitated DNA in 100ul ddH2O. | *Resuspend the precipitated DNA in 100ul ddH2O. | ||
Set up S1 nuclease digestion | Set up S1 nuclease digestion | ||
MDA amplicon: | MDA amplicon: 20ul | ||
10X S1 nuclease buffer: | 10X S1 nuclease buffer: 5ul | ||
3M NaCl: | 3M NaCl: 5ul | ||
H2O: | H2O: 15ul | ||
S1 nuclease (Invitrogen): | S1 nuclease (Invitrogen, 10U/ul): 5ul | ||
37c 1h -> 2C -> Purified with Qiaquick columns. | 37c 1h -> 2C -> Purified with Qiaquick columns. | ||
Check the size of debranched DNA with 1% argarose gel. |
Revision as of 01:21, 30 January 2009
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.