Alice:LabNotes/2008-1-11: Difference between revisions
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(New page: <b>Experiment 1:Preparation of padlock library</b><br> Real time PCR setup:<br> {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"...) |
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==Experiment 1:Preparation of padlock library== | |||
Real time PCR setup: | *Real time PCR setup: | ||
{| border="1" cellpadding="5" cellspacing="0 | {| border="1" cellpadding="5" cellspacing="0" | ||
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| align="center" style="background:#f0f0f0;"|''' ''' | | align="center" style="background:#f0f0f0;"|''' ''' | ||
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| Total ||100uL ||1600uL | | Total ||100uL ||1600uL | ||
|} | |} | ||
94C 2min | 94C 2min | ||
20x (94C 30 sec, 58C 2 min, 72C 1 min)<br> | 20x (94C 30 sec, 58C 2 min, 72C 1 min)<br> | ||
72C 5 min | 72C 5 min | ||
incubate at 15C forever | incubate at 15C forever | ||
Note: the PCR is usually stopped 3 cycles after the curve goes to plateau. | Note: the PCR is usually stopped 3 cycles after the curve goes to plateau. | ||
Agrose gel result (1-12-08): | |||
[[Image:ZhangLab_2 2008-01-12 15hr 00min.jpeg]] | |||
The bright bands on the bottom are the ladders for both columns (3uL ladder, 1 uL TBE buffer, and 1 uL of loading dye), | |||
and the rest of the wells contains sample 1-16 (4uL sample and 1uL of loading dye).<br> | |||
The bands on the left column (first 8 samples) don't good intensity, which is possibly caused by not well-mixed PCR reagents | |||
in the tube. |
Latest revision as of 20:50, 11 December 2010
Experiment 1:Preparation of padlock library[edit]
- Real time PCR setup:
' | X16 | |||||
2X Taq master mix | 50 uL | 800uL | ||||
100 uM forward primer | 0.5uL | 8uL | ||||
100 uM reverse primer | 0.5uL | 8uL | ||||
SYBR Green (50x) | 0.5uL | 8uL | ||||
dH2O | 48.2 | 768 | ||||
Template (10uM) | 0.2 uL | 3.2uL | ||||
Total | 100uL | 1600uL |
94C 2min 20x (94C 30 sec, 58C 2 min, 72C 1 min)
72C 5 min incubate at 15C forever Note: the PCR is usually stopped 3 cycles after the curve goes to plateau.
Agrose gel result (1-12-08): File:ZhangLab 2 2008-01-12 15hr 00min.jpeg The bright bands on the bottom are the ladders for both columns (3uL ladder, 1 uL TBE buffer, and 1 uL of loading dye), and the rest of the wells contains sample 1-16 (4uL sample and 1uL of loading dye).
The bands on the left column (first 8 samples) don't good intensity, which is possibly caused by not well-mixed PCR reagents in the tube.