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Team:Hawaii/Meeting/2008-06-26
From 2008.igem.org
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Contents |
Agenda
- Last Two Weeks: Recap
- Krystle:
- Cryofreeze stocks
- Grace:
- Triparental Conjugation
- Plasmid Prep & plasmid preps gels verification
- Margaret:
- pRL1383a final design
- pRL1383a low copy plasmid prep
- Norman:
- Primer Design notes (subtle variations & Hawaii protocol)
- Inventory organization (& -80C freezer map for iGEM rack)
Minutes
Present: NW, KS, GK, MR, KLS, GP, SC
BioBrick Plasmid Verifications
:*Use Forward and Reverse Verification Primers to verify the BioBrick using PCR :*Sequence plasmid preps.
- After we get the 1383a parts,etc, we are going to try to do all the sequencing at one time
BioBrick Assembly
:*May want to add the rbs to the beginning of a primer you use for your coding region.
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
- MR will get the nucleotide sequence for the ends of the omega interposon and the restriction sites immediately outside/around it from SC.
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
- Run 10 minipreprs (2ml each) and use 50 ul to combine all of them together.
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
