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

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>Jie deng
(New page: ==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 mole ratio of suppressor oligo:...)
 
>Jie deng
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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 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:
((600nM*2.5)/97k)*100=1.5nM.
  ((600nM*2.5)/97k)*100=1.5nM.
I prepared the 100x suppressor oligo as 150nM/each, then dilute to 10x working mixture.
  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.
  I adjusted the concentration of oligoes according to their read abundance.


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 = 180ul.
  The final volume of mixture = (50uM*0.5ul)/150nM = 180ul.
So I take 0.5ul oligo (50uM)/each and added ddH2O to get the final volume of 180ul.
  So I take 0.5ul oligo (50uM)/each and added ddH2O to get the final volume of 180ul.


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;
F1-H1: take 3ul/oligo;
  F1-H1: take 3ul/oligo;
A2-H3: take 1ul/oligo;
  A2-H3: take 1ul/oligo;
A4-H12: take 0.5ul/oligo;
  A4-H12: take 0.5ul/oligo;
add ddH2O to get the final volume of 180ul.
  add ddH2O to get the final volume of 180ul.

Revision as of 23:02, 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 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 = 180ul.
 So I take 0.5ul oligo (50uM)/each and added ddH2O to get the final volume of 180ul.
 For cpg55A subset, I adjusted the ration according to the  abundance range.
 A1-E1: take 5ul/oligo;
 F1-H1: take 3ul/oligo;
 A2-H3: take 1ul/oligo;
 A4-H12: take 0.5ul/oligo;
 add ddH2O to get the final volume of 180ul.