"
Team:Georgia State/Notebook/august
From 2013.igem.org
< Back
Week 4: 6/3 to 6/7
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 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 digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.
Week 5: 6/10 to 6/14
We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
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.
Week 6: 6/17 – 6/21
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.
Week 7: 6/24 -6/30
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 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.
Week 4: 6/3 to 6/7
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 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 digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.
Week 5: 6/10 to 6/14
We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
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.
Week 6: 6/17 – 6/21
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.
Week 7: 6/24 -6/30
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 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.
