Matt:LabNotes/2013-6-4: Difference between revisions
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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=== | ===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 in 140ul | *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 in 140ul | ||
*Use gDNA as template/target | *Use gDNA 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 | *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> | 10^4 cells x 10^4 rolonies/cell = 10^8 rolonies in dish <br> | ||
10^8 rolonies / 4,000 genes = 2.5 x 10^4 rolonies/gene (targets) <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==== |
Revision as of 19:44, 5 June 2013
Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-22
Probe Capture Plan
- 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
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 | |