Team:Hawaii/Meeting/2008-06-26

Agenda

 * 1) Last Two Weeks: Recap
 * 2) * Krystle:
 * 3) * Cryofreeze stocks
 * 4) * Grace:
 * 5) * Triparental Conjugation
 * 6) * Plasmid Prep & plasmid preps gels verification
 * 7) * Margaret:
 * 8) * pRL1383a final design
 * 9) * pRL1383a low copy plasmid prep
 * 10) * Norman:
 * 11) * Primer Design notes (subtle variations & Hawaii protocol)
 * 12) * Inventory organization (& -80C freezer map for iGEM rack)

Minutes
Present: NW, KS, GK, MR, KLS, GP, SC

BioBrick Plasmid Verifications
:*Sequence plasmid preps.
 * After we get the 1383a parts,etc, we are going to try to do all the sequencing at one time

Lichenase Gene

 * Check the Culture Collelction to see if they have Clostridium Thermocellum
 * Contact Charlie O’Kellly about getting clostridium to get Lichenase gene

pRL1383a parts

 * MR will get the nucleotide sequence for the ends of the omega interposon and the restriction sites immediately outside/around it from SC.
 * Look for the restriction sites closest to the transcription terminators located at the ends of the interposon, they are probably Hindi sites

Primers/Synthesized

 * Primers were ordered on Monday of this week
 * Look over primers and figure out which primers have to be augmented
 * Check your parts’ sticky ends.
 * You run into problems if they both have a 5’ or both have a 3’ ends

Plasmid Prep

 * For the nanospec results, 280 is protein, 260 is DNA
 * Verify by gel electrophoresis
 * Run a pure sample, as well as 1:10 and 1:100 dilutions
 * Suggestion from SC and GP for redoing the pRL1383a plasmid prep:
 * Run 10 minipreprs (2ml each) and use 50 ul to combine all of them together.
 * Look for a plasmid w/ origin of rep next to antibx resistance gene.
 * PCR both of them out together (Put sites that will let you clone it into the multiple cloning site of pRL1383a.
 * Select for spec and amp, or whatever the antibx that you chose. (Whatever grows should have two antibiotic resistance.)
 * Look into RSK ori requires pi protein

Alternate Plans:

 * Turn rsf1010 into a biobrick vector and use that to do the signal sequence project.
 * Make primers, cut the unnecessary restriction sites out of the plasmid
 * We will synthesize the necessary sequences to make it a standard BioBrick vector(primer verification/restriction sites/ etc.) with

Action Items
MR: Recheck, possibly redesign, primers for partA

GK: Convert PRL1383 into a biobrick vector

KS: Work on a timeline w/ target deadlines for project

Coming Up

 * Ordering Deadline, June 29th, See Shopping List