Team:Yale/Notebook
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Revision as of 00:07, 17 October 2014
Lab Notebook |
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Outline of Our Project
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TroubleShooting (T7)Okay, so we’re currently having some issues with the transformations, so let’s take a step back and look at what can go wrong, and how we can fix them: Template generation: For T7 RNA pol+pZE21 with the cr/ta system, the hairpins on the primers limit the efficacy of PCR and Gibson assembly.
Solutions: First, we should screen the plasmids we have to check and see if they do in fact contain T7. Ariel has designed primers for the test already, and if necessary we can use the pZE21 with CAT as a control, since the band would be a significantly shorter length.
We can also try a different method of assembly: restriction enzyme digest (may be difficult because there are none that anneal EXACTLY to the spots we want, some are a few base pairs off), or deactivating CAT’s start codon and placing T7 a little ways downstream of the crRNA sequence, INSIDE CAT (which, without the start codon, would not be translated).
Transformation: We need to drop dialyze longer. Our bacteria doesn’t seem to like the high salt concentration. Do duplicate plates.
Quality control on plates: be sure to streak a non-transformed plate every time to make sure the antibiotic is working.
Transform into a hardier strain/one better suited for transformations?
Construct Idea: DOPA + Spider silk{{Template:Team:Yale2014/Templates/Footer}} |