Matt:LabNotes/2013-6-4
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22
20gap Probe Capture Plan[edit]
- From [1] and [2]: ~37.4 pmol (2.35 ug) in 140ul = 16.79ng/ul(Combined on 6-6-2013 into single tube)
- Use HAPMAP (ID C1) gDNA (87ng/ul) as template/target
- Use a probe:target ratio of 100:1
- Hosuk wants to use some circularized oligos to generate rolonies
- Hosuk said there are about 3,000 cells per dish, I will round up to 10,000 so there are extra oligos for multiple experiments
10^4 cells x 10^4 rolonies/cell = 10^8 rolonies in dish
If we assume 50% of targets in reaction can assist circularization we need 2*10^8 targets = 3.32*10^-16 mol
At 100:1 that means we need 3.32*10^-14 mol of probes = 2.56*10^-18 mol of probe sets
Therefore the reaction set up below should be sufficient
100:1 Probe:Target Calculations[edit]
Probe set size | 12964 | probes |
gDNA template | 300 | ng |
gDNA MW | 1.95E+12 | g/mol (3x10^9 bp x 650 Da/bp + 157.9 Da) |
template | 1.5385E-19 | mol |
probe sets (100:1) | 1.5385E-17 | mol |
probe MW (12,964 probes, 193nt) | 814190000 | g/mol (12,964 probes x (193 nt/probe x 325 Da/nt + 79 Da)) |
amount probe required | 1.25263E-08 | g |
12.5 | ng | |
16 x 20ul reactions:
Components | Volume | 16X Volume |
gDNA (87ng/ul) | 3.45 | 55.2 |
Probe | 0.745 | 11.92 |
H2O | 13.805 | 220.88 |
10X Ligase Buffer | 2 | 32 |
Total | 20 | 320 |
- 40ul of Mineral oil on top
- Ran thermacylcer program "Kun -> CpG"