Revision as of 05:57, 8 December 2009 by >Alan6017518
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 = 1.923uL
- 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*4069
Bis 5.6ng/uL 4069Probes 100nt
Since 1 base = 330Da (for single stranded DNA, 660Da for ds),
the concentration of probe is 5.6 ng/ul /(100x330) = 0.000169697 = 169.697nM
# 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 4069 / [(6x10^23)x (169.697*10^-9M)]*10^6 =5.328uL
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 6h ->
6 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