Team Members: Sanju Timilsina and Parul Sirohi
Project title: Over expression of E. coli Acetyl- CoA carboxylase (ACC)sub-unit accC in E.coli to enhance fatty acid accumulation for Bio-fuel production”.
On Tuesday 09/26, we ran our DNA sample on agarose gel for second time. This time we load both our undigested and digested by EcoR1 DNA 1 and DNA 2 samples. We used 100bp marker DNA to compare the size of our DNA samples http://openwetware.org/wiki/Image:Cropped_pic_1_copy_-_Copy.jpg. The output was satisfactory this time because the digested DNA sample showed fragmented bands which we didn't saw on our previous experiment. Our, digested sample were fragmented and the undigested sample were up in the gel due to it's huge molecular size http://openwetware.org/wiki/Image:Cropped_pic_1_copy_with_label_-_Copy.jpg. So, we decided to do PCR to amplify our gene on our next lab i.e. 09/27.
09/27, Thursday, we prepared our Primer solution, diluted it to make it's working concentration 10uM and prepared PCR samples. We, run one negative control without DNA, one positive control with DNA and one control without primers. Control primers and DNA was provided by our instructor to us. We ran 12 different samples with 3 controls at three different temperature(55 0C, 60 0C and 65 0C), to check the best temperature for our Primers to work. We took the temperature reference from our Primer melting temperature, given at Primer information sheet. We put or samples into the thermocycler for overnight and we will take the samples out of thermocycler and store it at -20 0C on 09/28, 10:00am. And ,in our next lab 10/02, we will do Agarose gel electrophorosis to check if we have our DNA amplified.