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Team:NTU-Singapore/Notebook/9 July 2008
From 2008.igem.org
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E7 Story continued... | E7 Story continued... | ||
*Ethidium Bromide Gel showed clean bands at 2k mark, took PCR direct to PCR purified to 30ul. | *Ethidium Bromide Gel showed clean bands at 2k mark, took PCR direct to PCR purified to 30ul. | ||
| - | **Gel run-smeary band.. | + | **Gel run-smeary band...What went wrong? |
*Trouble SHOOT SHOOT | *Trouble SHOOT SHOOT | ||
Revision as of 15:02, 9 July 2008
E7 Story continued...
- Ethidium Bromide Gel showed clean bands at 2k mark, took PCR direct to PCR purified to 30ul.
- Gel run-smeary band...What went wrong?
- Trouble SHOOT SHOOT
- Protocol different from 8 July
- Optimize Protocol
- Protocol different from 8 July
HOW? Identified potential causes of this problem as:
- Sequence of protocols ran - should we run a gel extraction before PCR purification? Or vice-versa?
- Loading amount during gel electrophoresis
- Ratio of loading dye to sample
THUS..
We ran a gel with the following lane contents for comparison.
AND WE FOUND ...that running a PCR purification AFTER gel extraction yielded the nicest (i.e. clearest, least smeary) band - yay! Best loading amount is 8microlitres and the loading dye issue is a non-issue.
T7ptag Story 2
PCR product obtained..Ran Gel with 4ul..Good clear band Ran gel to purify the rest..loaded with 10ul
