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Lab Journal |
Week 25 (16/6 - 22/6) |
Monday 16/6 |
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The lab crash course began. We talked a lot about the project goal and the way of getting there. The team is now on the same page and ready for a great summer of exciting and interesting work to begin. In the lab we learned where everything was located and learned how to run a PCR.
- Sarah
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Tuesday 17/6 |
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In the lab: The PCR from yeasterday didn't work, so they were repeated. This time 50 µL PCR reaction were prepared. Gel purification was performed on the PCR products and the final concentration was measured by nanodrop.
We digested the products by using restriction enzymes XbaI og SpeI. We detected that the plasmid was only linearized, meaning that only one restriction enzyme/ restriction site was functional.
We worked on the basics in the iGEM concept. We discussed the definitions of biobrick, basis part, composite part and deviced. We learned the "Standard Assembly Method", it's condition, uses and limitations.
We defined the conditions for our own device and started searching for the necessary parts in the iGEM registry
-Victoria |
Wednesday 18/6 |
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The XbaI enzyme didn't work to cut plasmid 161 tuesday and we wanted to find out what was wrong. We had different theories as to what went wrong tuesday:
- Maybe the XbaI didn't have enough time to cut the plasmid. We only gave it 5 minutes.
- Maybe the restriction site on plasmid 161 didn't work.
To find out which of these theories was right we made three experiments:
- We wanted to do the same as on tuesday, but allow the enzyme to work for 15 minutes instead of 5 minutes, in case this was our mistake.
- We wanted to use the same plasmid as tuesday but cut it with EcoRI to see if this restriction site worked.
- We wanted to try to cut a new plasmid (RFP) with the same enzyme for the same amount of time (5 minutes), to find out if the enzyme worked or not.
We ran these three experiments and also a control for each of the two plasmids on a PCR, and observed which plasmids where cut. We found out that something was wrong with the X restriction site of plasmid 161, since XbaI didn't cut plasmid 161 (not with 15 minutes either) but did in fact cut pRFP. Furthermore we found out that the E restriction site on plasmid 161 worked, since EcoRI was able to cut it.
Besides the worked in the lab, we learned to design primers for Standard Assembly Method
- Sarah
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Thursday 19/6 |
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To be written.
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Friday 20/6 |
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Team page and Notebook page added to the wiki.
- Sarah
We tried to construct the TetR without the LVA-tag, 3 PCRs were made.
PCR 1 consisted of template 2:2P, primer #3 and #4.
PCR 2 consisted of template from previous made PCR2 and primers #3 and #5.
PCR 3 consisted of template 2:24D, primer #6 and #7.
Standard operating procedure was used for creating the 3 reactions with 50µL.
Our gel was odd, which turned out to be caused by mixing two different gel mixs.
However, we were still able to cut out the bands anyhow , and they seemed to be okay.
The gels were purified as the SOPs described.
We had to stop our work here, since we lacked enzymes and were therefore not able to digest the PCR products.
- Martin
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Saturday 21/6 |
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All the pipettes were calibrated
Odor-free strain - Yale
Odor-free strain - iGEM
A K12 MG1655 strain was plated out on agar, to colonize overnight, in order for us to produce more plasmid later on.
- Martin
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Sunday 22/6 |
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A coloni from the overnight plate was transferred to a bulb with 10 µL LB and TSB buffer was prepared using the SOB
- Martin
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