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

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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||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 -> <BR>4 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