Jie:LabNotes/CpgSeq/2009-6-19: Difference between revisions
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50X SYBG I 0.4ul 3.6ul | 50X SYBG I 0.4ul 3.6ul | ||
H2O 31.6ul 286.2ul | H2O 31.6ul 286.2ul | ||
[[Image: 20090622_cpg97k_A subset_No09_1~7.jpg]]20090622_cpg97k_A subset_No09_1~7 | |||
[[Image: 20090622_cpg97k_B subset_No09_1~7.jpg]]20090622_cpg97k_B subset_No09_1~7 |
Revision as of 23:01, 22 June 2009
set up the suppressor oligo mixture
the average concentration of cpg97k probe is ~20ng/ul (~600nM).The volume of probe in a 10ul capture reaction is 2.5ul. The volume of suppressor oligo is 1ul/capture reaction. The mole ratio of suppressor oligo: probe is 100:1. So the suppressor oligo concentration in a 10ul capture reaction should be: ((600nM*2.5)/97k)*100=1.5nM. I prepared the 100x suppressor oligo as 150nM/each, then dilute to 10x working mixture. I adjusted the concentration of oligoes according to their read abundance.
For cpg55B subset, the read abundance range from 430-1673. The oligo(50uM) volume used is 0.5ul/each. The final volume of mixture = (50uM*0.5ul)/150nM = 167ul. So I take 0.5ul oligo (50uM)/each and added 162ul ddH2O to get the final volume of 167ul.
For cpg55A subset, I adjusted the ration according to the abundance range. A1-E1: take 5ul/oligo; (25ul) F1-H1: take 3ul/oligo; (9ul) A2-H3: take 1ul/oligo; (14ul) A4-H12: take 0.5ul/oligo; (63ul) add ddH2O to get the final volume of 167ul.
recapture the PGP samples with two cpg97k subsets
The probe set was prepared by Alan.
No | sample | sample concentration | 10xLigase buffer | template+cpg97k_A(25.6ng/ul_06/18) vol+suppress oligo+H2O | template+cpg97k_B(21ng/ul_06/18) vol+suppressor oligo+H2O |
09_1 | NA21687*1 062507 | 95.4ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_2 | NA21781*1 102307 | 120.2ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_3 | NA21833*1 101807 | 121.5ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_4 | NA21660*1 053007 | 120.3ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_5 | NA21731*1 101807 | 127.3ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_6 | NA21846*1 100407 | 151.3ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
09_7 | NA21070*1 100406 | 311.2ng/ul | 1ul | 2+2.5ul+1ul+3.5ul | 2+2.5ul+1ul+3.5ul |
positive control | Jurkat bis-gDNA 200ng/ul | 1ul | 1+1ul+1ul+6ul | 1+1ul+1ul+6ul | |
negative control | 0 | 1ul | 0+1ul+1ul+6ul | 0+1ul+1ul+6ul |
dNTP concentration 10mM(NEB)
ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x80 40ul 8ul 8ul 16ul 8ul
PCR
Template 10ul x9 2X iProof Mastermix 50ul 450ul AmpF6.3SoL (10uM) 4ul 36ul AmpR6.3SoL (10uM) 4ul 36ul 50X SYBG I 0.4ul 3.6ul H2O 31.6ul 286.2ul
File:20090622 cpg97k A subset No09 1~7.jpg20090622_cpg97k_A subset_No09_1~7
File:20090622 cpg97k B subset No09 1~7.jpg20090622_cpg97k_B subset_No09_1~7