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Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period4/Dailylog
From 2013.igem.org
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6/10/13 KK, KP We ran our PCR product on a gel - this was the product of the PCR reaction with the new, correct, CRO primers. Despite this, our gel showed one, thick band at the bottom of the gel, but no 300 base pair band. We streaked out TT9901 and TT9907 and TT25281 fresh from the stabs to use as template on our next attempt, and Dr. Grose is going to do it with us.
6/12/13 KK, KP Dr. Grose helped us prepare PCR again. We're running PCR with both a negative control AND a positive control - a template and primers that Dr. Grose proved yesterday work. We changed to annealing temperature to 69 C on our phusion program and we added DMSO, which we had never added before. DMSO helps to separate G-C rich sequences. We're learning to anticipate the lab work we'll need to do in the future. Today we also made the gel with which we'll check our products on Wednesday, and we purified 100 microL of the plasmid pIG12 at a concentration of 116 ng/microL. This is the plasmid into which we'll be cloning CRO. Redge sent us 8 hilarious sketches of Baxter Bacteria and his pet Vector. I'm thrilled that this children's book is going to be a lot of fun! I told Redge to being his illustrative work already.
6/14/13 KK, KP We showed, finally, a positive result for our PCR! We amplified CRO! We had limited time today, but we performed a PCR clean-up with a kit from Dr. Grose's lab, and then we set the restriction digest of CRO and of pIG12 with PST-1 and EcoR-1. I made a low-melt gel to use tomorrow, after which we'll perform the ligation.
6/17/13 KK, KP, When we ran CRO and our plasmid on the low melt gel, we saw a beautiful band where CRO should be, but no distinct band of the plasmid. So, we set up another digest reaction of the plasmid and we saved part of our low-melt gel to run the plasmid tomorrow. TUESDAY 6/18/ We cut out the plasmid from the low melt gel, ligated it with the insert, waited an hour and a half, and then did a transformation of the plasmid into E.Coli to grow a lot of it. The E.Coli plates should be ready tomorrow.
6/19/13 KK, KP Clarice returned today! The transformation of the ligated plasmid (pIG12 + CRO gene insert) was ready today. Our plates showed good results - there were few colonies and NO colonies on our control plate. We set up 8 colony PCR reactions and a control. We'll have the results by Friday.
6/21/13 KK, KP
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