Jie:LabNotes/CpgSeq/2009-6-19

From ZhangLabWiki
Jump to navigation Jump to search

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