Team:Georgia State/Notebook/july

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<b>Week 8: 7/1 – 7/5</b>
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<b>Week 4: 6/3 to 6/7</b>
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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.
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We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
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<b>Week 9: 7/8 -7/12</b>
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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.
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We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
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We also digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.  
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We also performed a midiprep on pGAPZαA + MCS linker + RFP, made and filtered sterilized 1L 10% glycerol, made LB agar for chloramphenicol plates, and prepared electrocompetent E. coli.
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<b>Week 5: 6/10 to 6/14</b>
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<b>Week 10: 7/15 – 7/20</b>
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We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
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We had problems with arching during Electrotransporation of E. coli so we switched to transforming cells chemically.
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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.
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<b>Week 6: 6/17 – 6/21</b>
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<b>Week 11: 7/23 – 7/27</b>
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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.  
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We realized that the RFP that we had inserted in pGAPZαA + pGAPZαA MCS linker last year was not the optimal version of RFP to be inserted in yeast so we transformed E. coli with the what we thought was the more optimal RFP, but we picked the RFP from the wrong kit plate.  
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<b>Week 7: 6/24 -6/30</b>
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<b>Week 12: 7/29 – 8/2</b>
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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.
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We transformed from Kit Plate 3 N12 RFP into E. coli and performed minipreps on an overnight culture from one of the transformed colonies.
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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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Revision as of 22:11, 22 September 2013

Georgia State Wiki

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Week 8: 7/1 – 7/5
We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.

Week 9: 7/8 -7/12
We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
We also performed a midiprep on pGAPZαA + MCS linker + RFP, made and filtered sterilized 1L 10% glycerol, made LB agar for chloramphenicol plates, and prepared electrocompetent E. coli.

Week 10: 7/15 – 7/20
We had problems with arching during Electrotransporation of E. coli so we switched to transforming cells chemically.

Week 11: 7/23 – 7/27
We realized that the RFP that we had inserted in pGAPZαA + pGAPZαA MCS linker last year was not the optimal version of RFP to be inserted in yeast so we transformed E. coli with the what we thought was the more optimal RFP, but we picked the RFP from the wrong kit plate.

Week 12: 7/29 – 8/2
We transformed from Kit Plate 3 N12 RFP into E. coli and performed minipreps on an overnight culture from one of the transformed colonies.

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