Jie:LabNotes/CpgSeq/2009-6-19: Difference between revisions

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[[Image: 20090622_cpg97k_B subset_No09_1~7.jpg]]20090622_cpg97k_B subset_No09_1~7
[[Image: 20090622_cpg97k_B subset_No09_1~7.jpg]]20090622_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

Revision as of 01:38, 23 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

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