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Team:Technion/12 September 2012
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Ilya
- Repeated plate reader for tac+RS+mCherry and tac+RBS+mCherry in pCP. Got similar results to previous experiments. Constitutive expression of mCherry, without any effect of theophylline concentrations. Moreover, the tac+RBS+mCherry gave lower readings than tac+RS+mCherry, in contrast to expectations and one previous experiment. After talking to Roee, we decided to summarize the results along with some calculations and assumptions for the failure of the riboswitch. We will still submit this part to the registry, along with all of our results.
- Prepared a 0.4% gel for the gibson attempts on the phage. Unfortunately, we didn't have time to do the experiments today.
- Began setting up the restrictions for characterizing the MCS plasmid. Shahar and Hila will finish the digestions and run the results on a gel.
Inbal
-Colony PCR for the whole part- pTetO+m-cherry+eT7+terminator within pSB1AK3 (let's call it In1, for my convenience)- I picked randomly 5 colonies, The PCR didn't yield any results.
-I PCRed again pTetO+m-cherry (for the T3 cloning), one sample did actually was amplified, but the other- with the same conditions and PCR and equipment, wasn't amplified (the wonder of science!).
- I minipreped In1 from yestreday and the concentration is 202.5 ng/ul.
- I redid a colony PCR for In1 (again picked 5 colonies by random) but didn't get any good results (not the expected band -3600bp).
Asaf
I need to insert the long Riboswitch+polymerase genes to Psb1c3 plasmid.I minipreped the strain that contain the Psb1c3 with the MCS.
I got good DNA concentrations (75.5-120.5 ng/ul).
I cut the Psb1c3 with the MCS with EcoRI and PstI restriction enzymes.
I ran the cut Psb1c3 on gel for extraction. I got the expected 2kb bands.
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Shahar cleaned the DNA from the gel I extracted.
Hila
- Crating glycerol stock for all the positive colonies with the fragments 2C, 3C, 5C, and 8C.
- Mini-prep for the pSB1C3_MCS and the plasmids with the fragments and preparing them for shipment to iGEM HQ.
- Last scan for positive colonies with fragments 1C, 4C, 6C and 7C. If no positive colonies will be found I will begin the cloning from the beginning.
- Gel purification for additional F4, F4* and F5 for Gibson.
- Restriction for the characterization of pSB1C3_MCS plasmid. It seems in the gel that we have a problem with the XbaI, however all the restriction enzymes gave us the wanted band (XbaI gave two bands: one identical to the uncut plasmid band and one identical to the rest of the enzymes restriction product…).
Lior
Me and Noa checked our parts again with IPTG in various concentrations and various elutions in 96 well plate.
The T7+ALP performed great and the absorbance rose with the rise in IPTG concentration.
The T7+xyIE part didn't performed good enough and the absorbance was the same in various concentrations.
Noa
Evgeni
Preparation of starters from colonies that have grown on 'rest' plate after yesterday's transformation.
Shahar
from some reason the PCR for N4 & T3 with pTETO prefix and sufix did not worked. did new PCR with temprature 57 & 58 degrees.
Gel purification for additional F4, F4* and F5 for Gibson.
Rachel
- 6 starters of pSB1AK3 (includs double terminators BBa_B0015).
