From 2012.igem.org
Team:Technion/14_June_2012
Ilya
Today I prepared a gel for the purification of our fusion PCR products. Then Asaf and I ran the complete amount of our reaction products, cut out the band and purified it from the gel. In the purification process, we repeated the elution step from the column twice into different eppendorfs, because we were concerned that we won't have enough product. The DNA concentration in the first elution was 17.8 ng/ul. The concentration in the second elution was 15 ng/ul. Apparently, a lot of the DNA remains on the column after the first elution step. From now on, we will repeat the elution step into the same eppendorf to collect as much DNA as possible.
Next, I set up the next PCR reaction, to add the restriction sites. We decided to dilute the eluted DNA to get a 4.45 ng/ul solution which we used for the reaction. I ran the reaction with the appropriate primers with a Ta of 69 degrees. I made a mistake calculating the length of the PCR product and set the extension time for too long (24 secs instead of ~15 secs). I think the products should still be good. I will run a gel on Sunday to check that. The correct expected length is 847bp. Then we can finally start our cloning steps.
I also ordered the riboswitch+T3 RNAP from genome compiler today. Omri was very nice when I called him and helped me with the order details.
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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