Team:NYC Wetware/Notebook/Week11

From 2011.igem.org

(Difference between revisions)

Latest revision as of 03:26, 28 September 2011

NYC-iGEM wetware

Miniprep last of E. coli genes, and made glycerol stocks. Require verification.

Successful amplification of biobrick insert using RFC primers. And first successful D. rad PCR

Full plate of D. rad amplifications. Gel shows 6 definite successes and 6 'maybe's.

PCR pBAD promoter(K206000) for insertion in front of RBS and for assembly in composite promoter-RBS biobrick. Also PCR'ed preexisting composite promoter-RBS biobricks with some succes (K081005 fail, J04500 success). PCR RBS with RFC primers and with Verification primers for composite part.

Inoculate large cultures of pSB1C3+RFP as backbone for our screening plasmid.

Miniprep large cultures of pSB1C3+RFP.

Digest pSB1C3 backbone with E+P. Digest RBS plasmid with E+X to insert pBAD in front of RBS. Digest RBS from RFC and Ver PCRs with X+P as downstream part for assmbly into pSB1C3 with pBAD. Digest pBAD (K20600) with E+S for use as upstream part in RBS plasmid pSB1A2 and in composite part in pSB1C3 backbone.

@@ Line 20: / Line 20: @@
 <br/>
+Miniprep last of E. coli genes, and made glycerol stocks. Require verification.
 <br/>
-Looking to clone RBS into plasmid pSB1A10 which contain RFP. Transformed pSB1A10 from initial distribution.
-Successful transformation with multiple red colonies. Inoculated pSB1A10. <br/>
 <br/>
-Miniprepped pSB1A10. Digested and ligated RBS's B0030 and B0034 into pSB1A10. Looking to transform tomorrow.<br/>
+Successful amplification of biobrick insert using RFC primers. And first successful D. rad PCR
 <br/>
-Transformed new screening plasmid, pSB1A10, with the RBSs ligated.
-Transformed two T. cruzi radioresistance genes. Hopefully, the same radioresistance that the plant enjoys can be conferred on E. coli through biobricks. <br/>
 <br/>
-Ran PCR and gel to verify E. coli biobrick transformants. Verification primers amplify gene insertion plus ~100 bp in either direction. Samples whose lengths match expected sizes are sent for Sager sequencing.<br/>
+Full plate of D. rad amplifications. Gel shows 6 definite successes and 6 'maybe's.
+<br/>
+<br/>
+PCR pBAD promoter(K206000) for insertion in front of RBS and for assembly in composite promoter-RBS biobrick. Also PCR'ed preexisting composite promoter-RBS biobricks with some succes (K081005 fail, J04500 success). PCR RBS with RFC primers and with Verification primers for composite part.
+<br/><br/>
+Inoculate large cultures of pSB1C3+RFP as backbone for our screening plasmid.
+<br/>
+<br/>
+Miniprep large cultures of pSB1C3+RFP.
+<br/><br/>
+Digest pSB1C3 backbone with E+P. Digest RBS plasmid with E+X to insert pBAD in front of RBS. Digest RBS from RFC and Ver PCRs with X+P as downstream part for assmbly into pSB1C3 with pBAD. Digest pBAD (K20600) with E+S for use as upstream part in RBS plasmid pSB1A2 and in composite part in pSB1C3 backbone.