Team:Georgia State/Notebook/july

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<h2>Alisha/Merhawi, Group A</h2>
 +
<u>Transformation using chemical competent cells</u><br /> 
 +
<b>7/18/13</b><br />
 +
Page 2 Merhawi's notebook<br />
 +
Concentration of pGAPzaB=5ng/ul<br />
 +
Competent cells from "AM 3/19/13" <br />
 +
<b>Protocol</b><br />
 +
1. Mix 2ul of pGAPzaB DNA with 40ul of chemically competent cells <br />
 +
2. Put on ice for 10 minutes <br />
 +
3. 42°C water bath for 30 sec<br />
 +
4. Put on ice for 10 minutes <br />
 +
5. Add 800ul SOC <br />
 +
6. Recover overnight in shaking incubator @ 37°C <br />
 +
7. Plate next day on selective media<br />
 +
8. After plating, let sit in gel side for 45 minutes before plating into incubator  <br />
 +
<br />
 +
<u>Miniprep of pGAPzaB</u><br />
 +
<b>7/19/13</b><br />
 +
Page 111 Alisha’s notebook<br />
 +
Overnight culture made by Reza 7/18/13<br />
 +
Axygen <br />
 +
<b>Protocol</b><br />
 +
1. Collect 4 ml of overnight LB culture. Centrifuge at 12,000xg for 1 min to pellet the bacteria. Decant or pipette off as much of the supernatant as possible. (repeated step four times taking 1 (1000ul) of culture)<br />
 +
2. Resupend the bacterial pellet in 250ul of Buffer S1 (in fridge) by vortexing until pellet is resupended in solution. <br />
 +
3. Add 250ul of Buffer S2, invert tubes 6 times. <br />
 +
4. Add 350ul of Buffer S3 inverting 8 times. Centrifuge at 12,000xg for 10 minutes. <br />
 +
5. Place a Miniprep column into an uncapped 2ml Microfuge tube. Transfer the clarified supernatant from step 4 into the Miniprep column. Centrifuge at 12,000 for 1 minute.<br />
 +
6. Add 500ul of Buffer W1 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.<br />
 +
7. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute. <br />
 +
8. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.<br />
 +
9. Centrifuge at 12,000xg for 1 minute.<br />
 +
10. Transfer the column into a clean 1.5ml Microfuge tube. Add 80ul of Eluent to the column and let stand for 1 minute at room temp. centrifuge at 12,000xg for 1 minute.<br />
 +
11. Store a -20°C in Merhawi’s box. <br /><br />
-
<b>Week 4: 6/3 to 6/7</b>
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<h2>Kristin/Lydia, Group C</h2>
 +
<b>Week 8: 7/1 – 7/5</b>
<br />
<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 continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
-
<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.
+
-
<br />
+
-
We also digested 50 µl of both pGAPZαB and pGAPZαC and purified the samples using DNA precipitation and gel isolation.  
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<br /><br />
<br /><br />
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<b>Week 5: 6/10 to 6/14</b>
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<b>Week 9: 7/8 -7/12</b>
<br />
<br />
-
We digested pGAPZαA with either EcoRI and PstI or EcoRI and Bgl II and ran the samples on a gel.
+
We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
<br />
<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 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.
<br /><br />
<br /><br />
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<b>Week 6: 6/17 – 6/21</b>
+
<b>Week 10: 7/15 – 7/20</b>
<br />
<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 had problems with arching during Electrotransporation of E. coli so we switched to transforming cells chemically.
<br /><br />
<br /><br />
-
<b>Week 7: 6/24 -6/30</b>
+
<b>Week 11: 7/23 – 7/27</b>
<br />
<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 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.  
-
<br />
+
<br /><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.
+
<b>Week 12: 7/29 – 8/2</b>
 +
<br />
 +
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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Latest revision as of 22:47, 22 September 2013

Georgia State Wiki

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Alisha/Merhawi, Group A

Transformation using chemical competent cells
7/18/13
Page 2 Merhawi's notebook
Concentration of pGAPzaB=5ng/ul
Competent cells from "AM 3/19/13"
Protocol
1. Mix 2ul of pGAPzaB DNA with 40ul of chemically competent cells
2. Put on ice for 10 minutes
3. 42°C water bath for 30 sec
4. Put on ice for 10 minutes
5. Add 800ul SOC
6. Recover overnight in shaking incubator @ 37°C
7. Plate next day on selective media
8. After plating, let sit in gel side for 45 minutes before plating into incubator

Miniprep of pGAPzaB
7/19/13
Page 111 Alisha’s notebook
Overnight culture made by Reza 7/18/13
Axygen
Protocol
1. Collect 4 ml of overnight LB culture. Centrifuge at 12,000xg for 1 min to pellet the bacteria. Decant or pipette off as much of the supernatant as possible. (repeated step four times taking 1 (1000ul) of culture)
2. Resupend the bacterial pellet in 250ul of Buffer S1 (in fridge) by vortexing until pellet is resupended in solution.
3. Add 250ul of Buffer S2, invert tubes 6 times.
4. Add 350ul of Buffer S3 inverting 8 times. Centrifuge at 12,000xg for 10 minutes.
5. Place a Miniprep column into an uncapped 2ml Microfuge tube. Transfer the clarified supernatant from step 4 into the Miniprep column. Centrifuge at 12,000 for 1 minute.
6. Add 500ul of Buffer W1 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
7. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
8. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
9. Centrifuge at 12,000xg for 1 minute.
10. Transfer the column into a clean 1.5ml Microfuge tube. Add 80ul of Eluent to the column and let stand for 1 minute at room temp. centrifuge at 12,000xg for 1 minute.
11. Store a -20°C in Merhawi’s box.

Kristin/Lydia, Group C

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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