840:153g:Projects/project1/2008/10/14

A Flower of a Different Color by "Emblazon"

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DNA Purication and Vector extraction A Flower of A Different Color The DNA group (Rajiv, Tiffany and Melissa) discused with Axel about the next step for the DNA PCR product and RNA extraction procedures. We came to a conclusion that the DNA PCR Product will be used to be sequenced. To do so we will have to purify the DNA PCR product using the verbal protocol from Axel on DNA purification. The procedure is as stated: Combine all PCR products that amplified, samples C5, C6, E5, E8, and E9 into one centrifuge tube. Next we added .6 times the volume of the PCR sample PEG with 2.5M NaCl. Then incubated at room temperature for 15 minutes and centrifuge for 30 minutes at 15,000X. the supernant was discarded and the clear pellet was brought back up to volume with 20 mircoliters of diH2O. To test the amount of actual DNA present in the sample we used Electrophoresis to test the sample. The gel showed that there was DNA in the sample. After a discussion with Axel, the conclusion was to allow the DNA sample to sit at room temperature until Thursday and redo the electrophoresis with a 5 microliter sample. We will also have to preform PCR on fresh DNA and analyze that as well. This is needed to have enough product that can be sent off to be sequenced. The vector group(Angie,Binu,Sushma,Diwash)performed three restriction digests for each plasmid sample using a different enzyme for each digest. The enzymes used were fast digest Spe1,normal EcoR1,and fast digest Xbal. The digest was done with three different enzymes to make sure at least one of the enzymes cut the plasmids properly, and to see if the problems we were encountering with electrophoresis of our DNA were caused by the fast digest EcoR1 we were previously using in the procedure. We ran electrophoresis of our digested plasmids and also of undigested plasmids to make sure our plasmids were in fact present in the samples. The bluescript plasmids and the plasmids psB1A3 and psB1A7 from glycerol stocks received from MIT looked to contain the correct number of base pairs (bluescript around 3,000bp, and 1A3/1A7 around 2,000bp). The other samples of psB1A3 and psB1A7 from Axel's stock (DB1.3) were not properly digested, so we are not going to work with these plasmids any further. To confirm that the bluescript plasmid, 1A3 and 1A7 are capable of being properly digested and contain the correct number of base pairs, we will repeat the restriction digest and electrophoresis of these plasmids next lab.