Team:Hawaii/Notebook/2008-07-22

= Things we did today =

Alkaline Lysis Mini-prep

 * Margaret
 * Plasmid prep of pSB1A2, pSB1A3, and pSB3K3: 3 1.5 mL preps of the first two, and 10 of the third because it has a lower copy number. Used the protocol from Dr. Callahan's lab ([]).
 * I had to skip the phenol/chloroform step, so after adding Rnase A and incubating for 20 minutes, I spun down again, collected the supernatant and then precipitated with isopropanol.
 * The write-up for this prep can be found here under the 7/23 attempt.


 * Grace
 * nir, pilA, slr-1, slr-2, GFP fusion
 * Added 1 &mu;l 10mg/ml RNAse to each plasmid prep after transferring supernatant; incubated at 55C for 90 min.

Glycerol Stock

 * Margaret


 * glycerol stock of pSB1A2, pSB1A3, and pSB3K3. These DB3.1 strains containing these plasmids can be found in the -80C with the other plasmids in E. coli.


 * Grace
 * nir, pilA, slr-1, slr-2, GFP fusion, BB-pRL1383a, BBa_E0026

pRL1383a insert Clean-up

 * Margaret
 * in preparation for constructing the biobrick form of pRL1383a, cleaned the PCR products: mob, rep, oriV, and the aadA region using the "Isopropanol Precipitation for PCR purification" protocol from openwetware.org


 * Cleaned the oriT construct using the Qiagen Minelute Gel Purification kit.

Made 80% glycerol stock solution

 * Grace

Organized plasmid preps

 * Grace


 * Determined DNA concentrations of all plasmid preps to date
 * Relabeled all tubes w/ plasmid (or BioBrick part), DNA concentration, date of prep, and initials of person who prepped
 * Posted more detailed plasmid prep information on private website

BioBrick assembly/subcloning

 * Grace


 * Extracted GFP, B0024, B0034 from gel using MiniElute Spin Kit from Qiagen
 * Checked concentration of parts using nanodrop

DNA concentration of parts extracted from agarose gel
 * Ligated parts
 * nir + B0034 (rbs)
 * GFP + B0024 (tt)
 * GFP fusion + B0024 (tt)
 * Incubated ligation reaction for 2 hours at room temperature
 * Transformed DH5&alpha; with ligated parts
 * Used 5 &mu;l ligation reaction for each transformation

= Discussion =

= Quote of the Day = "History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson"