Kun:LabNotes/ASE/2007-12-5: Difference between revisions
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(New page: ==Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007== one reaction 3 reactions Template: 5ul ...) |
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94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. | 94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold. | ||
[[Image: 2007-12-05-cDNA-CES22k-PCR-PAGE.png|300px]] | |||
==Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07== | |||
Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency. | |||
<b>Reference:</b> H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM. <br> | |||
<b>Template:</b> | |||
#12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each | |||
#11-26-07: BJ cDNA 1ul | |||
#11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each | |||
Two 50ul reactions for each template. | |||
one reaction 24 reactions | |||
Template: 1ul each | |||
10x Buffer: 5ul 120ul | |||
25mM MgCl2 3ul 72ul | |||
25mM dNTP 0.6ul 14.4ul | |||
100uM AmpV6F 0.2ul 4.8ul | |||
100uM AmpV6R 0.2ul 4.8ul | |||
iTaq 0.5ul 12ul | |||
50X SYBG I 0.4ul 9.6ul | |||
H2O 39ul 936ul | |||
94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Well / Set''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''C(t)''' | |||
| align="center" style="background:#f0f0f0;"|'''pM*ul''' | |||
| align="center" style="background:#f0f0f0;"|'''# molecules/ul''' | |||
|- | |||
| A2||H1(1)||10.43||0.033||19530 | |||
|- | |||
| B2||H1(1)||10.11||0.041||24888 | |||
|- | |||
| C2||H2(1)||10.55||0.030||17790 | |||
|- | |||
| D2||H2(1)||10.31||0.035||21276 | |||
|- | |||
| E2||Hues6(1)?||7.91||0.220||131760 | |||
|- | |||
| F2||Hues6(1)?||8.01||0.204||122580 | |||
|- | |||
| G2||BJ(1)?||8||0.206||123660 | |||
|- | |||
| A3||H1(2)||12.49||0.007||4075.8 | |||
|- | |||
| B3||H1(2)||12.03||0.010||5799.6 | |||
|- | |||
| C3||H2(2)||11.15||0.019||11262 | |||
|- | |||
| D3||H2(2)||12.08||0.009||5569.8 | |||
|- | |||
| E3||Hues6(2)||8.73||0.118||71040 | |||
|- | |||
| F3||Hues6(2)||8.97||0.098||59052 | |||
|- | |||
| G3||BJ(2)||12.75||0.006||3358.2 | |||
|- | |||
| H3||BJ(2)||12.28||0.008||4779 | |||
|- | |||
| | |||
|} | |||
<b>I'm not completely sure whether I added the right template to E2,F2,G2,H2.</b> |
Latest revision as of 17:51, 6 December 2007
Exp. 1 Perform PCR on the capturing reactions started on 12/03/2007[edit]
one reaction 3 reactions Template: 5ul each 10x Buffer: 9.5ul 28.5ul 25mM MgCl2 6ul 18ul 25mM dNTP 1.2ul 3.6ul 100uM AmpSolV6F2 0.4ul 1.2ul 100uM AmpSolV6R2 0.4ul 1.2ul Platinium Taq 1ul 3ul 50X SYBG I 0.8ul 2.4ul H2O 76.7ul 231ul
94C 3min -> 10 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72 1min) -> 72C 3min -> 15C hold.
File:2007-12-05-cDNA-CES22k-PCR-PAGE.png
Exp.2 Perform real-time PCR to quantify the efficiency of the capturing reactions started at 12-03-07 and 11-21-07[edit]
Per Billy's suggestion, I'm using a diluted Solexa library with known concentration as the reference to quantify the circularization efficiency.
Reference: H1 gDNA Solexa library (11-27-07), 20.6nM, series diluted to 100pM, 10pM, 1pM, 100fM.
Template:
- 12-03-07: H1 cDNA, H2 cDNA, Hues6 cDNA, 1ul each
- 11-26-07: BJ cDNA 1ul
- 11-21-07: H1 cDNA, H2 cDNA, Hues6 cDNA, BJ cDNA, 1ul each
Two 50ul reactions for each template.
one reaction 24 reactions Template: 1ul each 10x Buffer: 5ul 120ul 25mM MgCl2 3ul 72ul 25mM dNTP 0.6ul 14.4ul 100uM AmpV6F 0.2ul 4.8ul 100uM AmpV6R 0.2ul 4.8ul iTaq 0.5ul 12ul 50X SYBG I 0.4ul 9.6ul H2O 39ul 936ul
94C 3min -> 30 cycles of (94C 45sec -> 57C 1min -> 72C 1min) -> 72C 3min -> 15C hold.
Well / Set | Sample | C(t) | pM*ul | # molecules/ul |
A2 | H1(1) | 10.43 | 0.033 | 19530 |
B2 | H1(1) | 10.11 | 0.041 | 24888 |
C2 | H2(1) | 10.55 | 0.030 | 17790 |
D2 | H2(1) | 10.31 | 0.035 | 21276 |
E2 | Hues6(1)? | 7.91 | 0.220 | 131760 |
F2 | Hues6(1)? | 8.01 | 0.204 | 122580 |
G2 | BJ(1)? | 8 | 0.206 | 123660 |
A3 | H1(2) | 12.49 | 0.007 | 4075.8 |
B3 | H1(2) | 12.03 | 0.010 | 5799.6 |
C3 | H2(2) | 11.15 | 0.019 | 11262 |
D3 | H2(2) | 12.08 | 0.009 | 5569.8 |
E3 | Hues6(2) | 8.73 | 0.118 | 71040 |
F3 | Hues6(2) | 8.97 | 0.098 | 59052 |
G3 | BJ(2) | 12.75 | 0.006 | 3358.2 |
H3 | BJ(2) | 12.28 | 0.008 | 4779 |
I'm not completely sure whether I added the right template to E2,F2,G2,H2.