Matt:LabNotes/2013-6-4: Difference between revisions

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>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===
*Use the ~37.4 pmole of probes 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] to produce maximum number of circularized probes with U's incorporated for Ho Suk's experiments
*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
|-
|
|}


56 ug of gDNA
*40ul of Mineral oil on top
140 ul of Probes
*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"