Jie:LabNotes/CpgSeq/2009-6-19: Difference between revisions
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==set up the suppressor oligo== | ==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 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. | ||
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A4-H12: take 0.5ul/oligo; (63ul) | A4-H12: take 0.5ul/oligo; (63ul) | ||
add ddH2O to get the final volume of 167ul. | add ddH2O to get the final volume of 167ul. | ||
==recapture the PGP samples with two cpg97k subsets== | |||
{| border="1" cellpadding="5" cellspacing="0" align="center" | |||
|- | |||
| align="center" style="background:#f0f0f0;"|'''No ''' | |||
| align="center" style="background:#f0f0f0;"|'''sample ''' | |||
|align="center" style="background:#f0f0f0;"|'''sample concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''template+cpg97k_A(25.6ng/ul_06/18) vol+suppress oligo+H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''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 |
Revision as of 00:32, 20 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
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