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:...)
 
>Sam Chiang
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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 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:
((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 = 167ul.
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 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 180ul.
  add ddH2O to get the final volume of 167ul.
 
==recapture the PGP samples with two cpg97k subsets==
The probe set was prepared by Alan.
 
{| 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||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
 
[[Image: 20090622_cpg97k_A subset_No09_1~7.jpg| 20090622_cpg97k_A subset_No09_1~7.jpg]]20090622_cpg97k_A 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
 
mix the A and B subsets to 1:1 mole ratio.
send half of the samples to Billy Li on 09-06-24.
 
No09_1_Mix_cpg97k 2.35ng/ul, 6.0nM x 40ul
No09_2_Mix_cpg97k 2.19ng/ul, 5.8nM x 30ul                      
No09_3_Mix_cpg97k 1.53ng/ul, 4.1nM x 48ul
No09_4_Mix_cpg97k 2.37ng/ul, 6.3nM x 30ul          
No09_5_Mix_cpg97k 2.93ng/ul, 7.8nM x 25ul
No09_6_Mix_cpg97k 2.32ng/ul, 6.2nM x 40ul                      
No09_7_Mix_cpg97k 1.91ng/ul, 5.1nM x 30ul
 
I got the Covaris sheared samples back on 09-06-24. The image on the gel:
 
[[Image:20090624_samples after Covaris shearing.jpg]]NA21687*1 062507 (No09_1) after Covaris shearing
The sheared DNA were not used due to the possible defects of Covaris shearing in Billy's lab.
 
===redo PCR with V6.3NH2 primer(2009-07-24)===
  I redo the PCR with V6.3NH2 primer and 2x Master mix (NEB) and purified the amplicons with Qiaquick column.
No09_1_Mix_cpg97k 920ng, 100ul
No09_2_Mix_cpg97k 610ng, 100ul                      
No09_3_Mix_cpg97k 780ng, 100ul
No09_4_Mix_cpg97k 680ng, 100ul          
No09_5_Mix_cpg97k 720ng, 100ul
No09_6_Mix_cpg97k 1260ng, 100ul                      
No09_7_Mix_cpg97k 770ng, 100ul

Latest revision as of 22:12, 4 August 2009

set up the suppressor oligo mixture[edit]

 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[edit]

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[edit]

 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
mix the A and B subsets to 1:1 mole ratio. 
send half of the samples to Billy Li on 09-06-24.
No09_1_Mix_cpg97k	2.35ng/ul, 6.0nM x 40ul
No09_2_Mix_cpg97k	2.19ng/ul, 5.8nM x 30ul	                       
No09_3_Mix_cpg97k	1.53ng/ul, 4.1nM x 48ul
No09_4_Mix_cpg97k	2.37ng/ul, 6.3nM x 30ul	           
No09_5_Mix_cpg97k	2.93ng/ul, 7.8nM x 25ul
No09_6_Mix_cpg97k	2.32ng/ul, 6.2nM x 40ul	                       
No09_7_Mix_cpg97k	1.91ng/ul, 5.1nM x 30ul

I got the Covaris sheared samples back on 09-06-24. The image on the gel:

File:20090624 samples after Covaris shearing.jpgNA21687*1 062507 (No09_1) after Covaris shearing

The sheared DNA were not used due to the possible defects of Covaris shearing in Billy's lab.

redo PCR with V6.3NH2 primer(2009-07-24)[edit]

 I redo the PCR with V6.3NH2 primer and 2x Master mix (NEB) and purified the amplicons with Qiaquick column.
No09_1_Mix_cpg97k	920ng, 100ul
No09_2_Mix_cpg97k	610ng, 100ul	                       
No09_3_Mix_cpg97k	780ng, 100ul
No09_4_Mix_cpg97k	680ng, 100ul	           
No09_5_Mix_cpg97k	720ng, 100ul
No09_6_Mix_cpg97k	1260ng, 100ul	                       
No09_7_Mix_cpg97k	770ng, 100ul