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> -->
100 x 2.5 x 10^4 = 2.5 x 10^6 probe sets => 4.151 x 10^-18 mol of probe sets
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

  • From [1] and [2]: ~37.4 pmol in 140ul
  • Use gDNA 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

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