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	[[Boston_UniversityPrimerDesign | Primer Design]]		[[Boston_UniversityPrimerDesign | Primer Design]]
-	== Week's (Ambitious) Goals ==	+	[[Boston_UniversityCompTransf | Making Shewanella Competent and Transforming Plasmid]]
-		+
-	Week of 6/4:	+
-		+
-	1. Evaluate the transformation that was done on Friday.	+
-		+
-	2. Confirm the correct plasmid (pJQ200)	+
-		+
-	3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.	+
-		+
-	4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.	+
-		+
-	5. Order primers.	+
-		+
-	6. Practice regular (non-error prone) PCR with the primers to check that they work.	+
-		+
-	7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.	+
-		+
-	8. Conjugate this plasmid into Shewy.	+
	== Materials We Need ==		== Materials We Need ==
-	Error-Prone PCR:  Need to Buy	+	Error-Prone PCR:  From CAB(?)
-	Ligases:  Need to Buy?	+	Ligases:  Need to Buy
	== Short-Term To-Do List ==		== Short-Term To-Do List ==
		+	EDIT THE WIIIIKIIII
-	Ordering of Error-Prone PCR Materials:  Not completed	+	== Protocols ==
		+	[[Boston_UniversityHeatShockProtocol | Calcium Chloride/Heat Shock Plasmid Transformations Protocol]]
-	Thank-You Letters sent to Pfizer:  Not Completed	+	[[Boston_UniversityFilterConjugationProtocol | Filter Cojugation Protocol]]
-	Thank-You Letters sent to BU ppl:  Not completed	+	[[Boston_UniversityTOPO | pTrcHis TOPO TA Expression Kit Cloning Protocol]]
-	== Protocols ==	+	[[Boston_UniversityNEBCutter| Using NEBCutter for checking specific restriction enzymes against a sequence]]
-	[[Boston_UniversityHeatShockProtocol | "Calcium Chloride/Heat Shock Plasmid Transformations"]]	+
-	== Question and Answer ==	+	[[Boston_UniversityGel | Making an Electrophoresis Gel]]
-	Can we get the kanR gene with appropriate sticky ends for BsaI and Tth111I? (DB)	+	[[Boston_UniversityZymo/SOB | Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
		+	]]
-	yeah, we can get the DNA template for the kanR gene from the lab.  we'll need to design some primers to PCR amplify it out with the BsaI and Tth111I cut sites.  after cutting with the restriction enzymes, the kanR will have the appropriate sticky ends.  frank suggested that we try ligating the kanR gene to the global regulator gene. (ie. kanR ligated to hlyU) and then stick that into the pJQ200.  this way we can select for succesfull hlyU transformations with kanamycin	+	== Question and Answer ==
-		+
-	Ok, good.  Do those primers still need to be ordered?  And if we follow Frank's suggestion, what will go in place of the gtmR gene?	+
-		+
-	== Relevant Publications and Links==	+
-	http://www.shewybase.bu.edu	+
-	https://www.atcc.org/common/catalog/numSearch/numResults.cfm?collection=mb-vector&atccNum=77482
	[https://2007.igem.org/Boston_University Back]		[https://2007.igem.org/Boston_University Back]

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Latest revision as of 16:43, 6 July 2007

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Contents 1 What We've Accomplished 2 Materials We Need 3 Short-Term To-Do List 4 Protocols 5 Question and Answer

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Making Shewanella Competent and Transforming Plasmid

Materials We Need

Error-Prone PCR: From CAB(?)

Ligases: Need to Buy

Short-Term To-Do List

EDIT THE WIIIIKIIII

Protocols

Calcium Chloride/Heat Shock Plasmid Transformations Protocol

Filter Cojugation Protocol

pTrcHis TOPO TA Expression Kit Cloning Protocol

Using NEBCutter for checking specific restriction enzymes against a sequence

Making an Electrophoresis Gel

Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB

Question and Answer

Back