IGEM:Virginia/2012/Notebook/Genetically engineered bacteriophage for diagnosis of whooping cough/2012/08/12: Difference between revisions
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|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Week 12</span> | ||
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== | ==(8/12 – 8/18)== | ||
We found too much contamination after the nanodrop. There were too many types | |||
of plasmids and therefore our preps were unusable. | |||
<br> | |||
Planned to transform BL21 pLyss E.coli with commercial T7 genome, our T7 | |||
genome, pET30a, and negative control (no DNA). | |||
<br> | |||
We took O.D of transformants to check if T7 had assembled and is lysing cells. | |||
<br> | |||
We also took some time to think about all that we had been doing. We needed to find | |||
out if the plasmids on the gel were actually the plasmids of interest. We planned that | |||
if PCR worked, we would re-transform and plate a larger volume. Then we would | |||
pick a single colony and proceed from there. Additionally, we would use BL21 only | |||
if we did not have to re-ligate. We would only use DH5-alpha if we did have to re- | |||
ligate. If PCR fails, we would do ligation. | |||
<br> | |||
After the cultures grew overnight, there was no growth for BL21 on LB/Kan. Lawn | |||
on BL21 + commercial T7 genome, and the individual colonies on BL21+77 genome. | |||
We transformed plasmids and BPP genome. First we found primers to PCR-amplify | |||
various genes of interest. | |||
<br> | |||
After performing gel electrophoresis, we found that the plasmids do not appear to | |||
contain the inserts. We therefore had to redo ligation and transformation. | |||
We tried unfolding and refolding the hCG-beta control protein and also tested | |||
pregnancy kits before and after. | |||
<br> | |||
We ran an experiment with DTT and a new pregnancy test, “New Choice.” We hoped | |||
to see the positive band appear after refolding of hCG-beta for DTT that left a faint | |||
band after unfolding. This shows that would show that our folding solution, when | |||
titrated, was successful. | |||
<br> | |||
We determined specifics for unfolding conditions and folding conditions for hCG- | |||
beta. | |||
<br> | |||
Ultimately, we were able to detect hCG with the pregnancy kit. We were able to | |||
break disulfide bonds so that the kit could not detect hCG, and then we remade the | |||
disulfide bonds so that the hCG was detectable again by the pregnancy kit. | |||
We also performed electrotransformation of RB50. | |||
Revision as of 17:25, 3 October 2012
Week 12 | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
(8/12 – 8/18)We found too much contamination after the nanodrop. There were too many types
of plasmids and therefore our preps were unusable.
|