Jie:LabNotes/CpgSeq/2009-6-19
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set up the suppressor oligo mixture[edit]
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[edit]
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[edit]
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
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
Egel purify the A subset captured targets. qiaquick purify the B subset captured targets. Gel quantification:
No09_1_A: 1.6 ng/ul No09_1_B: 4.6 No09_2_A: 1.57 No09_2_B: 3.42 No09_3_A: 0.94 No09_3_B: 4.47 No09_4_A: 2.42 No09_4_B: 2.31 No09_5_A: 2.31 No09_5_B: 3.85 No09_6_A: 1.48 No09_6_B: 4.85 No09_7_A: 1.43 No09_7_B: 2.87
mix the A and B subsets to 1:1 mole ratio. send half of the samples to Billy Li on 09-06-24.
No09_1_Mix_cpg97k 2.35ng/ul, 6.0nM x 40ul No09_2_Mix_cpg97k 2.19ng/ul, 5.8nM x 30ul No09_3_Mix_cpg97k 1.53ng/ul, 4.1nM x 48ul No09_4_Mix_cpg97k 2.37ng/ul, 6.3nM x 30ul No09_5_Mix_cpg97k 2.93ng/ul, 7.8nM x 25ul No09_6_Mix_cpg97k 2.32ng/ul, 6.2nM x 40ul No09_7_Mix_cpg97k 1.91ng/ul, 5.1nM x 30ul
I got the Covaris sheared samples back on 09-06-24. The image on the gel:
File:20090624 samples after Covaris shearing.jpgNA21687*1 062507 (No09_1) after Covaris shearing
The sheared DNA were not used due to the possible defects of Covaris shearing in Billy's lab.
redo PCR with V6.3NH2 primer(2009-07-24)[edit]
I redo the PCR with V6.3NH2 primer and 2x Master mix (NEB) and purified the amplicons with Qiaquick column. No09_1_Mix_cpg97k 920ng, 100ul No09_2_Mix_cpg97k 610ng, 100ul No09_3_Mix_cpg97k 780ng, 100ul No09_4_Mix_cpg97k 680ng, 100ul No09_5_Mix_cpg97k 720ng, 100ul No09_6_Mix_cpg97k 1260ng, 100ul No09_7_Mix_cpg97k 770ng, 100ul