Team:NTU-Singapore/Notebook/9 July 2008

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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

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. 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

PCR product obtained..Ran Gel with 4ul..Good clear band Ran gel to purify the rest..loaded with 10ul

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