Team:Hawaii/Notebook/2008-08-14

= Things we did today =

Colony PCR

 * Margaret


 * 5 colonies of rep, P1 lytic region, and aadA(pRL1383a) region, each. pSB1A3 was used as a control.
 * I changed the annealing time to 3min 30 seconds instead of the extension time, so the sites were not amplified properly. I ran the aadA(pRL1383a) construct on a gel and got a few bands, none of which were the right size. One of them should have been right though, so I am assuming this ligation did not work.
 * I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.


 * Krystle
 * Krystle update

Culture

 * Margaret
 * Plasmid preps from yesterday did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
 * Krystle
 * Inoculated for plasmid prep of GFPf and J04430 tomorrow.

Restriction Digest

 * Margaret


 * digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.


 * Grace


 * Checked Krystle's RE digests from last night on a gel
 * Also ran J33207 PCR product on same gel
 * Excised J33207 and GFPf bands from gel
 * RE digested: [[Image:081408REdigests.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.]]
 * J33207, J04430, BB-pRL1383a with EcoRI and PstI
 * pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
 * BB-pRL1383a with HindIII and BamHI (from lab -20C)
 * Ran RE digests on a gel to purify segments of interest
 * Also ran RE digested B0015 from Krystle

Gel extraction

 * Krystle


 * Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel

Sequencing

 * Margaret


 * Verified sequence of oriT, inserted into pSB1A2. The sequences of oriT and the BioBrick sites are correct.

Oligonucleotide Design

 * Margaret & Grace


 * We are having trouble with TT, RBS and Promoter ligations, so we decided to synthesize these parts.
 * B0016, I14032, B0030, & B0034

= Discussion =
 * Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.

= Quote of the Day = "Happy chickens taste better. - KSGSK"