Team:Hawaii/Notebook/2008-07-29

= Things we did today =

Gel purification of ccdB PCR product

 * Grace

RE digests

 * Grace


 * Digested pRL1383a (from last night) with BamHI
 * Digested B0030 and B0024 (from last night) with EcoRI
 * Sequentially digested gel purified ccdB with HindIII and BamHI

Construction of BioBricks

 * Grace


 * Purified RE digests on EtBr stained 1.2% agarose gel
 * Extracted bands for B0030 and B0024
 * Other lanes FAILED!
 * SYBR Safe doesn't show up well, even under UV (arg...)

Direct Band Extraction Method

 * Norman
 * make two comb gels
 * run test UPA band along with ladder
 * attempt to pipette out DNA as it runs into the second well
 * no more gel cutting???

PCR amplification of pRL1383a parts

 * Margaret


 * Made a reaction buffer containing {38.5ul, 20ul accusure, 10ul dNTPs, & 5ul 5XBuffer}. This should be good for 20 50ul reactions.


 * Amplified with Accusure: aadA (BBa_J23012), aadaA (pRL1383a), rep region, oriV
 * Amplified with Green Taq: rep region


 * On 7-30 I ran a gel.

Media

 * Margaret
 * started to make LB amp100 plates, but autoclave room is closed. Remember to do it tomorrow.

Plasmid prep

 * Krystle
 * Innoculated LB+amp with pRL1383a harboring E. coli (from cryostock)

PCR

 * Krystle
 * PCR reactions to isolate GFP, GFPf, nir from plasmid

= Discussion =  SYBR Safe
 * Blue light still doesn't illuminate the bands well. No bands (not even the ladder) are visible. Resolution *much* better with short wave UV. EtBr better bet for now?

Restriction enzymes
 * HindIII and BamHI in the lab -20C freezer (in box labeled "TKW Restriction Enzymes") fail. They're really old and don't appear to cut well anymore. Digestion of pRL1383a resulted in a long smear.

= Quote of the Day = LOL! http://www.youtube.com/watch?v=J0s0Y3-BCaw