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

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==Capturing System Setup==                       
==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
  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

Revision as of 05:57, 8 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 = 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