Jie:LabNotes/CpgSeq/2009-6-19: Difference between revisions
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==set up the suppressor oligo== | ==set up the suppressor oligo== | ||
the average concentration of cpg97k probe is ~20ng/ul (~600nM).The volume of probe in a 10ul capture reaction is 2.5ul. | 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. | The mole ratio of suppressor oligo: probe is 100:1. | ||
So the suppressor oligo concentration in a 10ul capture reaction should be: | So the suppressor oligo concentration in a 10ul capture reaction should be: | ||
Line 10: | Line 10: | ||
For cpg55B subset, the read abundance range from 430-1673. | For cpg55B subset, the read abundance range from 430-1673. | ||
The oligo(50uM) volume used is 0.5ul/each. | The oligo(50uM) volume used is 0.5ul/each. | ||
The final volume of mixture = (50uM*0.5ul)/150nM = | The final volume of mixture = (50uM*0.5ul)/150nM = 167ul. | ||
So I take 0.5ul oligo (50uM)/each and added ddH2O to get the final volume of | 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. | For cpg55A subset, I adjusted the ration according to the abundance range. | ||
A1-E1: take 5ul/oligo; | A1-E1: take 5ul/oligo; (25ul) | ||
F1-H1: take 3ul/oligo; | F1-H1: take 3ul/oligo; (9ul) | ||
A2-H3: take 1ul/oligo; | A2-H3: take 1ul/oligo; (14ul) | ||
A4-H12: take 0.5ul/oligo; | A4-H12: take 0.5ul/oligo; (63ul) | ||
add ddH2O to get the final volume of | add ddH2O to get the final volume of 167ul. |
Revision as of 23:15, 19 June 2009
set up the suppressor oligo
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.