Team:Georgia State/Notebook/august

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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.

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 <a href="https://2013.igem.org/Team:Georgia_State/Notebook"><b>< Back</b></a><br />
+<b>Week 13: 8/5 - 8/9</b>
-<b>Week 4: 6/3 to 6/7</b>
 <br />
-The plates made during Week 3 of transformed E.coli + pGAPZαB + MCS Linker and E. coli + pGAPZαC + MCS Linker showed very few colonies. We picked one and grew an overnight culture.  We plated 200 µl of the cultures from and performed minipreps on the remaining sample. We digested these minipreps with EcoRI and XbaI and also used the same enzymes to digest minipreps of pGAPZαC that we had made in earlier weeks for comparison. We ran the samples on a 1% agarose gel at 80V for 60 minutes but only the pGAPZαC sample showed DNA.
+The minipreps from the week before had low DNA concentrations so we used gel purification to clean the samples.
 <br />
-We digested pGAPZαC with Eco RI to test our ligases and performed a DNA precipitation of the digest. We ran 20 µl of the samples on a gel at 80 V for 90 minutes and excised the DNA fragment. After gel isolating the linearized bands we ligated with either the ligase made by New England Biolabs or the ligase made by Promega Corp. We used the samples to transform E. coli and plated on low salt LB + Zeocin plates.
+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.
 <br />
-We also digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.
+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.
 <br /><br />
-<b>Week 5: 6/10 to 6/14</b>
+<b>Week 14: 8/12 - 8/16</b>
 <br />
-We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
+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.
 <br />
-We made and overnight culture from plates containing pGAPZαC + MCS linker that we had made last week. Half of the sample was used to perform minipreps and the other half was extracted by Phenol Chloraform. Half of the samples were digested with Bgl II and Kpn I and the other half with Bgl II and Spe I.
+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.
+<br />
+We also digested pGAPZαC+ Linker using EcoRi and PstI.  Then we gel isolated the sample to increase the purity of the sample.
 <br /><br />
-<b>Week 6: 6/17 – 6/21</b>
+<b>Week 15: 8/19 - 8/23</b>
 <br />
-We made another overnight culture from plates containing pGAPZαC + MCS linker from Week 4 and performed minipreps on them. We digested those minipreps and minipreps that had been prepared last week with either Bgl II and Kpn I or Bgl II and Spe I. We ran the digestions on a gel at 80 V for 90 minutes.
+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.
+<br />
+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.
+<br />
+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.
 <br /><br />
-<b>Week 7: 6/24 -6/30</b>
+<b>Week 16: 8/26 - 8/30</b>
 <br />
-We ran multiple double digestions on pGAPZαC and pGAPZαC + pGAPZαC MCS linker to verity that they didn’t contain any foreign DNA. First using Eco RI and BamH I, BamHI and Bgl II, and Pst and Bgl II, and then added a double digestion with Kpn I and Bgl IIbut where unable to confirm that the pGAPZαC MCS linker had been successfully transformed into E. coli.
+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.
 <br />
-We continued digesting the pGAPZαC plasmid and the pGAPZαC MCS linker, ligating them, and performing transformations while varying incubation temperature and times in order to optimize our protocol.
+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.
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