Team:Hawaii/Meeting/2008-08- 7

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Agenda

Present: Dr. Presting, Dr. Gautz, Dr. Callahan, Norman, Krystle, Grace, & Margaret

Concerning the Omega Interposon: We have had problems amplifying, so Dr. C suggested that we make it into a BioBrick by simply digesting a BB plasmid with one of the sites also in the polylinker of the Omega Interposon and neatly shoving it in that vector. Preliminary knowledge of what is available makes me want to put O.I. into the ccdB gene. I need to find out if this gene will be disrupted. If this doesn't work, Norman suggested that we synthesize an insert to put it in. For example BglII could be the restriction site engineered into this insert, it is compatible with BamHI, so when the O.I. is inserted, the site will be destroyed.
Concerning Ligation:

We need to use at least 100ng-1ug of insert
Ethanol Precipitation, from Norman's experience, yields 10 fold less DNA than you start with.
You must clean up the PCR reaction before digest, the taq will keep adding nucleotides if you do not.
We should start using better controls for the transformation of our ligation products. As a negative control, use the vector ligated back to itself.

Check to see if you will need IPTG in addition to X-Gal. Does the strain of E. coli produce the repressor? Is the repressor site on the operon?
solubilize the X-Gal in dimethylformamide (it is not soluble in aqueous solutions). Add the IPTG and X-Gal separately as together they will precipitate and not go into the agar.

TB is good for over-night cultures. This broth yields 2x more cells and unlike LB, does not leave a pile of dead cells at the bottom of the tube. This broth does not work for transformations.

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]