Team:Hawaii/Meeting/2008-06-26

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Agenda

Last Two Weeks: Recap
- Krystle:
1. - Cryofreeze stocks
- Grace:
1. - Triparental Conjugation
  - Plasmid Prep & plasmid preps gels verification
- Margaret:
1. - pRL1383a final design
  - pRL1383a low copy plasmid prep
- Norman:
1. - 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

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

<Person A>: Task

Coming Up

Ordering Deadline, June 29th, [http://packrat.stjohn.hawaii.edu/prestinglab/wiki/Shopping_List See Shopping List]

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

@@ Line 19: / Line 19: @@
 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
+===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 ==