Team:NTU-Singapore/Notebook/9 July 2008

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Revision as of 10:38, 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..S*** What went wrong?

Trouble SHOOT SHOOT
- Protocol different from 8 July
  - Optimize Protocol

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

@@ Line 6: / Line 6: @@
 **Protocol different from 8 July
 *** Optimize Protocol
-HOW?
-Take Gel extracted E7(50ul) direct, Gel extract(50ul)-PCR Purified to run gel to see the difference..To see the effectiveness of PCR purification and Gel Extract..Compare!
+'''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.
+[[Image:Protocol_Optimization_NTU.png]]
+'''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