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Team:Georgia State/Notebook/august
From 2013.igem.org
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Week 13: 8/5 - 8/9
The minipreps from the week before had low DNA concentrations so we used gel purification to clean the samples.
We transformed E. coli with the RFP from Kit Plate 3, 8L and plated it. We also plated some of the pGAPZαA + Unknown RFP for comparison.
We found a stop codon in the middle of the cDNA of the Mambalgin I protein that we had designed. We removed the stop codon and change the cDNA so that it would fall in the reading frame of pGAPZαC instead of pGAPZαB, since we were closer to standardizing pGAPZαC. We sent the new sequence to IDT labs.
Week 14: 8/12 - 8/16
We ligated two samples of pGAPZαC + Linker with the new Mambalgin I with two different ligases. We used chemical transformation of samples, and let the samples recover in SOC broth overnight in the 37°C shaking incubator. Then plated the samples on selective media plates the next day.
The transformed E. coli cells with pGAPZαC + Linker + Mambalgin I grow so we performed minipreps on an overnight culture. The we digested the minipreps using Bgl II and Xba I, Eco RI and Pst I, and Avr II. We ran the samples in a 0.8% agarose gel at 70 V for 110 minutes.
We also digested pGAPZαC+ Linker using EcoRi and PstI. Then we gel isolated the sample to increase the purity of the sample.
Week 15: 8/19 - 8/23
We digested samples of pGAPZαC + Linker + Mambalgin I and pGAPZαC + Linker with the same enzymes to verify the insertion of the Mambalgin I cDNA. We performed double digestions using EcoRI and PSt I, Bgl II and Xbal, Bgl II and BamHI, and did a triple digestion using Bgl II, Xbal, and BamHI.
We also performed whole cell PCRs of E. coli with the pGAPZαC + Linker + Mambalgin I and ran all the samples on the same gel. We ran the samples on a .8% agarose gel at 30 V for 1010 minutes.
The colony PCRs had a large concentration of DNA around 200 bp that was unexpected so reran the colony PCRs but recieved the same strange ban around 200 bp.
Week 16: 8/26 - 8/30
We ran colony PCRs on the pGAPZαC + Linker + Mambalgin I using a different set of forward and reverse primers, but continued to have unexpected bands.
Since the digestions had verified the insertion of the Mambalgin I cDNA, we linearized minipreps with Avr II. We performed an ethanol precipitation on the samples, then isolated the cDNA by running it on a .8% agarose gel at 15V for 999 minutes. We then gel purified the sample.
Week 13: 8/5 - 8/9
The minipreps from the week before had low DNA concentrations so we used gel purification to clean the samples.
We transformed E. coli with the RFP from Kit Plate 3, 8L and plated it. We also plated some of the pGAPZαA + Unknown RFP for comparison.
We found a stop codon in the middle of the cDNA of the Mambalgin I protein that we had designed. We removed the stop codon and change the cDNA so that it would fall in the reading frame of pGAPZαC instead of pGAPZαB, since we were closer to standardizing pGAPZαC. We sent the new sequence to IDT labs.
Week 14: 8/12 - 8/16
We ligated two samples of pGAPZαC + Linker with the new Mambalgin I with two different ligases. We used chemical transformation of samples, and let the samples recover in SOC broth overnight in the 37°C shaking incubator. Then plated the samples on selective media plates the next day.
The transformed E. coli cells with pGAPZαC + Linker + Mambalgin I grow so we performed minipreps on an overnight culture. The we digested the minipreps using Bgl II and Xba I, Eco RI and Pst I, and Avr II. We ran the samples in a 0.8% agarose gel at 70 V for 110 minutes.
We also digested pGAPZαC+ Linker using EcoRi and PstI. Then we gel isolated the sample to increase the purity of the sample.
Week 15: 8/19 - 8/23
We digested samples of pGAPZαC + Linker + Mambalgin I and pGAPZαC + Linker with the same enzymes to verify the insertion of the Mambalgin I cDNA. We performed double digestions using EcoRI and PSt I, Bgl II and Xbal, Bgl II and BamHI, and did a triple digestion using Bgl II, Xbal, and BamHI.
We also performed whole cell PCRs of E. coli with the pGAPZαC + Linker + Mambalgin I and ran all the samples on the same gel. We ran the samples on a .8% agarose gel at 30 V for 1010 minutes.
The colony PCRs had a large concentration of DNA around 200 bp that was unexpected so reran the colony PCRs but recieved the same strange ban around 200 bp.
Week 16: 8/26 - 8/30
We ran colony PCRs on the pGAPZαC + Linker + Mambalgin I using a different set of forward and reverse primers, but continued to have unexpected bands.
Since the digestions had verified the insertion of the Mambalgin I cDNA, we linearized minipreps with Avr II. We performed an ethanol precipitation on the samples, then isolated the cDNA by running it on a .8% agarose gel at 15V for 999 minutes. We then gel purified the sample.
