Difference between revisions of "Team:MIT/pdestmcherry"

Latest revision as of 05:58, 8 October 2016

I NEEDED A PAGE!!!!!!!

During the insertion of the the pENTR constructs into the pDEST, the mCherry gets cut out of the plasmid. When transformed into eColi on plates containing Ampicillin, the mCherry acts as a negative screening tool for choosing colonies that contain the properly-assembled plasmid of choice. the pDEST plasmid here is designed with the express purpose of distribution for use by other teams and scientists.

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Using the pDEST with mCherry is extremely similar to normal ligation reaction, transformations, and plating protocols.

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Many thanks are owed to the Tufts iGEM 2016 team, for agreeing to test our construct for us!

Materials

Promoter pENTR plasmid: L4-Promoter-R1 working concentration 5 fmol/μL

Gene pENTR plasmid: L1-Gene-L2 working concentration 5 fmol/μL

pDEST:mCherry plasmid working concentration 10 fmol/μL

Nuclease-free TE

200μL strip tube for each reaction

5x LR Clonase II

Proteinase K

Procedure

Into each tube: </li> SUBLISTS?????????

1 µl of the promoter pENTR

1 µl of the gene pENTR

1 µl of the pDEST-mCherry

Add 1 µl of TE </li> Add 1 µl of LR clonase (kept in -80)</li> Cap tubes, flick to mix and pulse-spin to collect liquid at the bottom</li> Incubate between 12-24 hours at room temperature. </li>

Proteinase K-kill

Add 1 μL proteinase K to each tube Flick tubes to mix and pulse-spin Incubate at 37° for 15 minutes, or room temperature for an hour. Can be used in transformation or stored at -20° indefinitely

@@ Line 28: / Line 28: @@
 Cap tubes, flick to mix and pulse-spin to collect liquid at the bottom</li>
 Incubate between 12-24 hours at room temperature.  </li>
+</p>
+<p> <h5>Proteinase K-kill</h5>
+Add 1 μL proteinase K to each tube
+Flick tubes to mix and pulse-spin
+Incubate at 37° for 15 minutes, or room temperature for an hour.
+Can be used in transformation or stored at -20° indefinitely
 </p>