Jie:LabNotes/CpgSeq/2009-6-19

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Revision as of 00:32, 20 June 2009 by >Jie deng
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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

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 2+5ul+2ul

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 
                                                          x50  25ul       5ul      5ul       10ul      5ul