Team:Georgia State/Notebook/september

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Week 17: 9/2 - 9/6
We used the electrically component P. pastoris cells, that we had made in the second week of the competition, and transformed them with pGAPZαC + Linker + Mambalgin I. We plated the cells on YDPs + Zeocin plates.
We also linearized more of the pGAPZαC + Linker + Mambalgin with Avr II and purified the sample through gel isolation and purification in case our original transformation did not work.

Week 18: 9/8 - 9/13
We purified samples of the pGAPZαC + Linker and pGAPZαC +Linker + Mambalgin I through gel isolation and purification. We reran our confirmation digestions from 8/19/13 in a 1% agarose gel at 15 V for 999 minutes to get an improved picture of the gel.
We performed PCRs on the P. pastoris with pGAPZαC + Linker + Mambalgin I and ran on a 1% gel at 70 V for 120 minutes.
We ran an SDS page to recover the Mambalgin I protein but did not recover any proteins in our gel.

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 <a href="https://2013.igem.org/Team:Georgia_State/Notebook"><b>< Back</b></a><br />
+<b>Week 17: 9/2 - 9/6</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.
+We used the electrically component P. pastoris cells, that we had made in the second week of the competition, and transformed them with pGAPZαC + Linker + Mambalgin I. We plated the cells on YDPs + Zeocin plates.
 <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 also linearized more of the pGAPZαC + Linker + Mambalgin with Avr II and purified the sample through gel isolation and purification in case our original transformation did not work.
-<br />
-We also digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.
 <br /><br />
-<b>Week 5: 6/10 to 6/14</b>
+<b>Week 18: 9/8 - 9/13</b>
 <br />
-We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
+We purified samples of the pGAPZαC + Linker and pGAPZαC +Linker + Mambalgin I through gel isolation and purification. We reran our confirmation digestions from 8/19/13 in a 1% agarose gel at 15 V for 999 minutes to get an improved picture of the gel.
 <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.
+We performed PCRs on the P. pastoris with pGAPZαC + Linker + Mambalgin I and ran on a 1% gel at 70 V for 120 minutes.
-<br /><br />
-<b>Week 6: 6/17 – 6/21</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 ran an SDS page to recover the Mambalgin I protein but did not recover any proteins in our gel.
-<br /><br />
-<b>Week 7: 6/24 -6/30</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.
-<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.
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