Team:WashU/Week8

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Revision as of 18:50, 18 July 2012

Monday, July 16

We ran multiple digests today, following the standard biobrick assembly protocol.

Digestions
Z and C: digest with P and E
Ligation 2: digest with X and P
Ligation 2: digest with X
Z and C: digest with E
ZCD: digest with X and P
UGTCs2: digest with X and P
2: digest with P
2: digest with B

The gel of these digests can be shown below.

In addition, we ran several PCRs today. Following the NEB protocol, we ran four PCRs to amplify the CCD plasmid (PUT PROPER NAME HERE) and also tried four colony PCRs, using colonies 2, 4, 13 and ______.

To enable amplification of our plasmid PSL2131, PsbA2, and CS42S, we designed primers to multiply the aforementioned DNA pieces.

We have started cultures of E. coli doubly transformed with our Z construct and CS42S at different chloramphenicol concentrations. We will miniprep them after they grow to obtain the DNA.
Tuesday, July 17

We repeated the digests and PCRs from yesterday in order to ascertain what went wrong. The PCRs yielded primer dimers and were thus unsuccessful, so we plan on troubleshooting our procedure to determine why our reactions are failing. [PICTURE OF GEL]

In order to increase our output of carotenoids in Synechocystis, we began a PAL mutant liquid culture of the cyanobacterium in order to transform with in the next few days.

Wednesday, July 18
This morning, we miniprepped a culture of PC42, our ligation of plasmid PSL2131 and construct CS42S. Then, we ran a gel of double and single digests of PC42, cut with X and P and just P, along with an uncut PC42 as a control. [GEL SHOWN BELOW]

In addition, we took the cultures of the double digest from Monday, miniprepped them, nanodropped, and then digested them with EcoRI and PstI. The digestions were then run on a gel [PICTURE BELOW]

Finally, in order to test whether our old culture of double transformation of E. coli were still maintaining their plasmids, we miniprepped some of the culture, digested, and ran the resultant on a gel. [PICTURE]

Thursday, July 19

Friday, July 20

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 To enable amplification of our plasmid PSL2131, PsbA2, and CS42S, we designed primers to multiply the aforementioned DNA pieces.
+We have started cultures of <i>E. coli</i> doubly transformed with our Z construct and CS42S at different chloramphenicol concentrations. We will miniprep them after they grow to obtain the DNA.
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+This morning, we miniprepped a culture of PC42, our ligation of plasmid PSL2131 and construct CS42S. Then, we ran a gel of double and single digests of PC42, cut with X and P and just P, along with an uncut PC42 as a control. [GEL SHOWN BELOW]
+In addition, we took the cultures of the double digest from Monday, miniprepped them, nanodropped, and then digested them with EcoRI and PstI. The digestions were then run on a gel [PICTURE BELOW]
+Finally, in order to test whether our old culture of double transformation of <i>E. coli</i> were still maintaining their plasmids, we miniprepped some of the culture, digested, and ran the resultant on a gel. [PICTURE]
 <br>