AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
(Removing all content from page)
 
Line 1: Line 1:
===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
{| {{table}}
| 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==
{| {{table}}
| Capturing System||NA12878||NA18507||NA20431||Jurkat (New)||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||5.808||1.043||13.219||13.561||15.248
|-
| Volume of 10X Ampligase buffer||2.500||2.500||2.500||2.500||2.500
|-
| 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 -> <BR>6 cycels (95C 1min -> 55C for  4h),add 2ul more SLN mix after 1st cycle -> 95C 5min -> <BR>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

Latest revision as of 06:32, 8 December 2009