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
|
|