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Revision as of 03:48, 4 October 2012
Wet Lab - June
-
Week 4
Having finished our bootcamp, we started working in our lab space in Weill Hall. This week, we began our work to construct our salicylate reporter plasmids using a sensing region including the transcriptional activator nahR. We also continued our work with the Gibson mutagenesis of the nah operon (which degrades naphthalene to salicylate) by attempting to confirm successful construction. We also began our adventure in attempting to transform into Shewanella via electroporation. Daily Details
-
Week 4
June 27th, Wednesday
This morning, Steven made glycerol stocks of the 3 Gibson colonies grown overnight. He then did minipreps on the 3 Gibson colonies followed by double digests using PstI and KpnI to check whether the Gibson procedure was successful. Around noon, Steven and Dylan attempted the Myers and Myers Shewanella Electroporation with p17c (pSB3C5). Dylan and Swati then set up a Phusion PCR to amplify the salicylate-sensing region out of BBa_K228227 (to eventually control the expression of mtrB), before running a gel of the restriction digests of the Gibson products. However, our ladder ran strangely, and the gel was determined to be inconclusive in showing that our Gibson colonies appeared to be successful. That night, Spencer and Steven set up overnight cultures of JG700, nahR, and the 3 Gibson colonies.
June 28th, Thursday
This morning, Dylan and Caleb did minipreps of all the cultures from the day before. They then set up a gel of Phusion PCR of nahR from Wednesday. That afternoon, Swati set up a transformation of p14k into DH5a cells. The cells were recovered for 1 hour at 37C in LB before plating. Dylan set up sequencing of the Gibson products with our newly designed primers while Shweta gel extracted the nahR from the gel set up by Dylan and Caleb. After Shweta’s gel extraction, Swati set up a double digestion of the product with EcoRI and AscI. That night, Steven ran a gel of the nahR digest while Spencer set up overnight cultures of p14k, p16k, pSB3C5, and pBBRBB.
June 29th, Friday
In the morning, Dylan set up a Vent PCR to amplify p21a (salicylate sensing region) and his previous Phusion PCR band. That afternoon, he gel purified the PCR products and set up a double digestion with EcoRI-HF and AscI. Spencer miniprepped the overnight cultures from Thursday. Later that night, Dylan set up another Phusion PCR to amplify the nah operon from Gibson colony 1 and to append Biobrick cutsites into the product for future ligation into pSB3C5. Swati started the gel extraction of p21a.
June 30th, Saturday
Today, Dylan started out the day by running the PCR of the Gibson colony 1 on a gel, gel extracted the band, and set up another Phusion PCR with an extension time of 3 minutes to get more of the construct. That afternoon, Swati finished her p21a gel extraction and dephosphorylated pBBRBB+mtrB for ligation with p21a. Swati desalted the ligation on Millipore membrane paper and electroporated her ligation into DH5a. After letting the cells recover in LB for 1 hour, Swati plated her ligations. Later that night, Steven and Spencer set up two transformations into PNNL electrocompetent Shewanella one at a voltage of 0.75 kV, and the other at 0.55 kV prescribed by Myers and Myers.
-
Week 4
Having finished our bootcamp, we started working in our lab space in Weill Hall. This week, we began our work to construct our salicylate reporter plasmids using a sensing region including the transcriptional activator nahR. We also continued our work with the Gibson mutagenesis of the nah operon (which degrades naphthalene to salicylate) by attempting to confirm successful construction. We also began our adventure in attempting to transform into Shewanella via electroporation.Daily Details:
June 27th, Wednesday
This morning, Steven made glycerol stocks of the 3 Gibson colonies grown overnight. He then did minipreps on the 3 Gibson colonies followed by double digests using PstI and KpnI to check whether the Gibson procedure was successful. Around noon, Steven and Dylan attempted the Myers and Myers Shewanella Electroporation with p17c (pSB3C5). Dylan and Swati then set up a Phusion PCR to amplify the salicylate-sensing region out of BBa_K228227 (to eventually control the expression of mtrB), before running a gel of the restriction digests of the Gibson products. However, our ladder ran strangely, and the gel was determined to be inconclusive in showing that our Gibson colonies appeared to be successful. That night, Spencer and Steven set up overnight cultures of JG700, nahR, and the 3 Gibson colonies.
June 28th, Thursday
This morning, Dylan and Caleb did minipreps of all the cultures from the day before. They then set up a gel of Phusion PCR of nahR from Wednesday. That afternoon, Swati set up a transformation of p14k into DH5a cells. The cells were recovered for 1 hour at 37C in LB before plating. Dylan set up sequencing of the Gibson products with our newly designed primers while Shweta gel extracted the nahR from the gel set up by Dylan and Caleb. After Shweta’s gel extraction, Swati set up a double digestion of the product with EcoRI and AscI. That night, Steven ran a gel of the nahR digest while Spencer set up overnight cultures of p14k, p16k, pSB3C5, and pBBRBB.
June 29th, Friday
In the morning, Dylan set up a Vent PCR to amplify p21a (salicylate sensing region) and his previous Phusion PCR band. That afternoon, he gel purified the PCR products and set up a double digestion with EcoRI-HF and AscI. Spencer miniprepped the overnight cultures from Thursday. Later that night, Dylan set up another Phusion PCR to amplify the nah operon from Gibson colony 1 and to append Biobrick cutsites into the product for future ligation into pSB3C5. Swati started the gel extraction of p21a.
June 30th, Saturday
Today, Dylan started out the day by running the PCR of the Gibson colony 1 on a gel, gel extracted the band, and set up another Phusion PCR with an extension time of 3 minutes to get more of the construct. That afternoon, Swati finished her p21a gel extraction and dephosphorylated pBBRBB+mtrB for ligation with p21a. Swati desalted the ligation on Millipore membrane paper and electroporated her ligation into DH5a. After letting the cells recover in LB for 1 hour, Swati plated her ligations. Later that night, Steven and Spencer set up two transformations into PNNL electrocompetent Shewanella one at a voltage of 0.75 kV, and the other at 0.55 kV prescribed by Myers and Myers.
