AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 5: Difference between revisions
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Probe:target ratio 200:1 (according to Alice, 100:1 works too) | Probe:target ratio 200:1 (according to Alice, 100:1 works too) | ||
*Billy uses 250ng | *Billy uses 250ng | ||
Old Jurkat Template(104ng/uL) | |||
Use 200ng total, so = 2uL | Use 200ng total, so = 2uL | ||
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==Setup Capturing Reaction== | ==Setup Capturing Reaction== | ||
{| {{table}} | {| {{table}} |
Revision as of 05:01, 6 December 2009
Calculation
Probe:target ratio 200:1 (according to Alice, 100:1 works too) *Billy uses 250ng Old Jurkat Template(104ng/uL) Use 200ng total, so = 2uL
- One genome mass = 3pg
- First calculate the # of genome your target represents
- For 200ng, it represents 200ng/3pg=66666.67 genomes
- You want to have 200x the probes as the target 200ng/3pg*200
- You want every single probe to target ratio to be 200:1 200ng/3pg*200*55,000
Ms_Meth 22ng/uL 55,000Probes 100nt Since 1 base = 330Da (for single stranded DNA, 660Da for ds), the concentration of probe is 22 ng/ul /(100x330) = 0.000666667 = 666.67nM # of molecules per mole = 6X10^23 # of probes needed/ concentration of probes = Volume of probes needed the volume the probe needed is 200ng/3pg x 200 x 55,000 / [(6x10^23)x (666.67*10^-9M)]*10^6 =1.83uL
Calculation for Jurkat
1 Base-ssDNA (Da) | 330.000 |
1 Base-dsDNA (Da) | 660.000 |
One genome mass (pg) | 3.000 |
# of genome your target represents | |
# of molecules per mole | 6.00E+023 |
Concentration of Probes (nM) | 16.970 |
Min. template input (ng) | 100.000 |
Template Input (ng) | 200.000 |
Probes to target ratio (X to 1) | 200.000 |
Probe Concentration (ng/ul) | 0.560 |
Number of probes | 4069.000 |
Number of NT in probes | 100.000 |
Template Concentration (ng/ul) | 104.000 |
Volume of probes needed | 5.328 |
Setup Capturing Reaction
Capturing System | NA12878 | NA18507 | NA20431 | Jurkat | Jurkat (Old) |
Volume of probes | 5.328 | 5.328 | 5.328 | 5.328 | 5.328 |
Volume of templates | 11.364 | 16.129 | 3.953 | 3.610 | 1.923 |
Volume of H20 | 6.808 | 2.043 | 14.219 | 14.561 | 16.248 |
Volume of 10X Ampligase buffer | 1.500 | 1.500 | 1.500 | 1.500 | 1.500 |
Total Volume | 25.000 | 25.000 | 25.000 | 25.000 | 25.000 |
Capturing System Setup
95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h ->
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold