Matt:LabNotes/2013-6-4: Difference between revisions
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>Mzcai (Created page with "Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22 ===Probe Capture=== *Use the ~37.4 pmole of probes from [http://genome-tech.ucsd.edu/La...") |
>Mzcai |
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22 | Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22 | ||
===Probe Capture=== | ===20gap Probe Capture Plan=== | ||
* | *From [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22] and [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-29]: ~37.4 pmol (2.35 ug) in 140ul = 16.79ng/ul(Combined on 6-6-2013 into single tube) | ||
*Use gDNA as template/target | *Use HAPMAP (ID C1) gDNA (87ng/ul) as template/target | ||
*Use a probe:target ratio of 100:1 | *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 <br> | |||
<!-- not necessary b/c Hosuk only wants for RCA: 10^8 rolonies / 4,000 genes = 2.5 x 10^4 rolonies/gene (targets) <br> --> | |||
If we assume 50% of targets in reaction can assist circularization we need 2*10^8 targets = 3.32*10^-16 mol <br> | |||
At 100:1 that means we need 3.32*10^-14 mol of probes = 2.56*10^-18 mol of probe sets <br> | |||
Therefore the reaction set up below should be sufficient | |||
====100:1 Probe:Target Calculations==== | ====100:1 Probe:Target Calculations==== | ||
{| {{table}} | |||
| 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: | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''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" |
Latest revision as of 20:44, 5 December 2013
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"