Kun:LabNotes/ASE/2007-12-9: Difference between revisions

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(New page: ==Exp. 1 Perform CES22k eSNP capture on PGP and hybrid samples== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | ali...)
 
 
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==Exp. 1 Perform CES22k eSNP capture on PGP and hybrid samples==
==Exp. 1 Perform CES22k eSNP capture on PGP and hybrid samples==
Genomic targets: 800ng/3pg per haploid genome = 2.67x10^5 copies = 2.22x10~-19 moles
Probes: 2.22x10~-19 moles * 22,000 * 125 = 1.22x10^-12 moles.


{| border="1" cellpadding="5" cellspacing="0" align="center"
{| border="1" cellpadding="5" cellspacing="0" align="center"
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|2||GM20431 cDNA (121ng/ul)||0.8ul||1.5ul||9ul||2.2ul
|2||GM20431 cDNA (121ng/ul)||0.8ul||1.5ul||9ul||2.2ul
|-
|-
|3||GC1F cDNA(48ng/ul)||2ul||1ul||9ul||1ul
|3||GC1F cDNA(48ng/ul)||2ul||1.5ul||9ul||1ul
|-
|-
|4||GC1EP cDNA(52ng/ul)||2ul||1ul||9ul||1ul
|4||GC1EP cDNA(52ng/ul)||2ul||1.5ul||9ul||1ul
|-
|-
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|''' '''
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| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
|-
|-
|5||Hues6 gDNA(113ng/ul)||5ul||1.5ul||7ul||0ul
|5||Hues6 gDNA(113ng/ul)||5ul||1.5ul||5ul||2ul
|-
|-
|6||Hues6 cDNA(155ng/ul)||0.7ul||1.5ul||7ul||4.3ul
|6||Hues6 cDNA(155ng/ul)||0.7ul||1.5ul||5ul||6.3ul
|-
|-
|7||H1 gDNA(222ng/ul)||3.6ul||1.5ul||7ul||0ul
|7||H1 gDNA(222ng/ul)||3.6ul||1.5ul||5ul||3ul
|-
|-
|8||H1 cDNA(35ng/ul)||3ul||1.5ul||7ul||3ul
|8||H1 cDNA(48ng/ul)||3ul||1.5ul||5ul||5ul
|-
|-
|9||H2 gDNA(200ng/ul)||4ul||1.5ul||7ul||0ul
|9||H2 gDNA(200ng/ul)||4ul||1.5ul||5ul||2ul
|-
|-
|10||H2 cDNA(48ng/ul)||2ul||1.5ul||7ul||3ul
|10||H2 cDNA(35ng/ul)||3ul||1.5ul||5ul||5ul
|-
|-
|11||BJ cDNA(18ng/ul)||5ul||1.5ul||7ul||0ul
|11||BJ cDNA(18ng/ul)||5ul||1.5ul||5ul||2ul
|}
|}


94C 5min->65C 40h-> add 1.5ul SLN mix (1U/ul stoffel, 1U/ul AmpLigase, 100uM dNTP) -> 65C 1h -> 40 cycles of (95C 1min -> 65C 5min) -> 37C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.
94C 5min->65C 40h-> add 1.5ul SLN mix (1U/ul stoffel, 1U/ul AmpLigase, 100uM dNTP) -> 65C 1h -> 40 cycles of (94C 2min -> 65C 15min) -> 37C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.
 
<b>Note:</b> I decided to terminate this experiment after the discussion with Billy regarding to some new modification of the protocol. I took the tubes out from the thermocycler prior to adding SLN mix, and stored them at -20C.
 
<b>2007-12-11:</b> I restarted the reactions with a modified protocol (use 60C instead of 65C).
 
94C 5min->60C 46h-> add 1.5ul SLN mix (2U/ul stoffel, 1U/ul AmpLigase, 200nM dNTP) -> 60C 1h -> 40 cycles of (94C 2min -> 650C 15min) -> 30C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.

Latest revision as of 18:55, 4 January 2008

Exp. 1 Perform CES22k eSNP capture on PGP and hybrid samples[edit]

Genomic targets: 800ng/3pg per haploid genome = 2.67x10^5 copies = 2.22x10~-19 moles Probes: 2.22x10~-19 moles * 22,000 * 125 = 1.22x10^-12 moles.

Template 10x buffer CES22k probes #5 (127nM) H2O
1 GM20431 gDNA(347ng/ul) 2.3ul 1.5ul 9ul 0.7ul
2 GM20431 cDNA (121ng/ul) 0.8ul 1.5ul 9ul 2.2ul
3 GC1F cDNA(48ng/ul) 2ul 1.5ul 9ul 1ul
4 GC1EP cDNA(52ng/ul) 2ul 1.5ul 9ul 1ul
Template 10x buffer CES22k probes #4 (177nM) H2O
5 Hues6 gDNA(113ng/ul) 5ul 1.5ul 5ul 2ul
6 Hues6 cDNA(155ng/ul) 0.7ul 1.5ul 5ul 6.3ul
7 H1 gDNA(222ng/ul) 3.6ul 1.5ul 5ul 3ul
8 H1 cDNA(48ng/ul) 3ul 1.5ul 5ul 5ul
9 H2 gDNA(200ng/ul) 4ul 1.5ul 5ul 2ul
10 H2 cDNA(35ng/ul) 3ul 1.5ul 5ul 5ul
11 BJ cDNA(18ng/ul) 5ul 1.5ul 5ul 2ul

94C 5min->65C 40h-> add 1.5ul SLN mix (1U/ul stoffel, 1U/ul AmpLigase, 100uM dNTP) -> 65C 1h -> 40 cycles of (94C 2min -> 65C 15min) -> 37C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.

Note: I decided to terminate this experiment after the discussion with Billy regarding to some new modification of the protocol. I took the tubes out from the thermocycler prior to adding SLN mix, and stored them at -20C.

2007-12-11: I restarted the reactions with a modified protocol (use 60C instead of 65C).

94C 5min->60C 46h-> add 1.5ul SLN mix (2U/ul stoffel, 1U/ul AmpLigase, 200nM dNTP) -> 60C 1h -> 40 cycles of (94C 2min -> 650C 15min) -> 30C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.