Janet B. Matsen:Closed Lab Questions

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Revision as of 08:23, 28 February 2012 by Janet B. Matsen (Talk | contribs)
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I define "closed" as "not bothering me incessantly." One can always learn more but we have to stop at a point otherwise we would never move forward with our research!

If you have any input/corrections/references please drop [User:Janet B. Matsen|me] a line.

Contents

Colony PCR

  • Q: The gel is dark where I loaded it -- what does this mean?
    • A: It probably means you overloaded the PCR with too many cells.

Primers/Oligos

Should I pay extra to further purify large bp oligos?

  • Usually no. The sequencing companies just want your money. Do sequence your creations to confirm that you made what you expected and remember you can always screen a new colony if the first one looks bad.

My primers are yellow!

  • Don't worry -- this is normal!
    normal oligos have a yellow color that is visible with large synthesis scales
    normal oligos have a yellow color that is visible with large synthesis scales

BioBricks

The parts in the registry have sequence that don't include the prefix & suffix... do they come with it?

  • Yes, they come with a prefix/suffix. You can find which BioBrick method a part is compatable with under the part's "Assembly Compatibility" listing. Most are RFC 10 (http://partsregistry.org/partsdb/scars.cgi), the standard format.

How do I decide the spacing between a promoter and ribosome binding site? A ribosome binding site & the start codon?

  • Recall that a promoter starts making a transcript at the +1 site, no matter the sequence. The -10 and -35 locations in the promoter that have large importance are numbered relative to this +1 site. You can have any arbitrary sequence before the RBS, and people often include operators for genetic regulation. Or, you can put the ribosome binding site next. This is more standard. The ribosome binding site needs to be correctly spaced from the start codon, and you have to look at the individual RBS to determine it. For some, a BioBrick scar is the perfect distance between the end of the RBS and the start codon; this is useful for making an expression vector like UW's 2010 iGEM vectors.
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