Janet B. Matsen:Closed Lab Questions
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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.
- 1 Lab Basics:
- 2 Colony PCR
- 3 Primers/Oligos
- 4 Cloning
- 5 Gene Expression & Plasmids
- 6 BioBricks
Why do we disinfect with 70% EtOH? Isn't 100% more potent?
- A: I have read that you don't want to denature the proteins that allow substances like water and ethanol into the cell. If you do, the EtOH can't get into the cytosol. By using 70%, you denature some of the transporter proteins but leave enough to allow more to enter the cell. Also, 70% is cheaper than 100%!
Wow.. these 2 batches of TB media are very different in color... did I mess up?
- A:Maybe, but not necessarily. Different batches of yeast and tryptone can have very different compositions. This is one of the purposes of defined media.
The gel is dark where I loaded it -- what does this mean?
- A: It probably means you overloaded the PCR with too many cells.
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!
How do I decide whether to gel purify after digestions (before ligations)
- The BioBrick/Ginkgo manual doesn't instruct you to. Besty always does.
- Um... I don't do much restriction enzyme cloning at this point... Gibson rules!
Gene Expression & Plasmids
=== Is a two plasmid system where the two plasmids have different antibiotic resistance genes but the same origin of replication stable? === (7/16/2012)
- A collaborator implied use of a different antibiotic is sufficient. However, I suppose this could lead to unpredictability in the number of each plasmid. For example, suppose the copy origin they share yields 100 plasmid copies. It could be that 10 copies of the antibiotic A & 90 copies of the antibiotic B plasmids is equally favorable as 90 copies of the antibiotic A plasmid & 10 of the antibiotic B plasmid. Presuming the two plasmids encode different proteins, the relative ratios of the expression levels would be drastically important.
- If allowed to compete in a flask, the burden of each plasmid will affect the distribution of the plasmids.
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, and that the promoter is only for transcription and the RBS is only for translation. The -10 and -35 locations in the promoter that are significant for binding 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.