Team:Hawaii/Notebook/2008-08-12

= Things we did today =

Test of lab's RE (cont.)

 * Grace
 * Ran RE digests from yesterday on a 1.2% agarose gel
 * BamHI and HindIII digested DNA not visualized. Too little DNA added?
 * NotI works. All plasmid preps except I14032 resulted in 2 bands of the correct size.
 * Note: There is genomic DNA in all preps (band that looks like fire up top)

Verified transformants

 * Grace


 * Colony PCR of transformants to check for correct insert
 * GFP+tt = unsuccessful
 * GFPf+tt colonies 1, 3, 5 good
 * slr1+GFPf colony 5 potentially good
 * pilA+GFPf colonies 1, 2, 3, 4 potentially good
 * Ligated slr, pilA with GFPf (ES digested) instead of GFPf (XP digested). Still got colonies, some which are potentially good. Hm...
 * Restreaked:
 * GFPf+tt colony 5
 * slr1+GFPf colony 5
 * pilA+GFPf colonies 1 and 4
 * Will colony PCR again tomorrow to prep for sequencing

3A assembly

 * Grace


 * Ligated p+r with g or s onto pSB1A2
 * pnir+rbs with GFP, slr1, pilA
 * plac+rbs with GFP, slr, pilA
 * Transformed into DH5&alpha; using 5 &mu;l ligation reaction

Rear ligation of signal sequences and GFPf

 * Grace


 * Ligated slr and pilA with XbaI and PstI digested GFPf
 * Transformed into DH5&alpha; using 5 &mu;l ligation reaction

[[Editing Team:Hawaii/Notebook/2008-08-11|Transformation
Margaret


 * Re-streaked yesterday's transformation
 * Colony PCR on each of the re-streaked colonies

Construction of Omega Interposon
Margaret


 * Digested pSMC121 and pSB1A2 with SmaI
 * Ligated parts together (over-night ligation)

Plasmid Prep
Margaret


 * B0015, B0030, E0040, J33207
 * Drying in hood

Glycerol Stock
Margaret


 * oriT (3 bottles) and I14032 (1 bottle)

Media Making
Margaret
 * Amp100 plates, Solution 3 for plasmid preps, 100mM IPTG(look in -20C with the antibiotics)

Sequencing

 * Grace


 * Downloaded sequences from CORE Hawaii. Began assembling contigs and checking sequences.

= Discussion =

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