840:153g:Projects/project2/2008/11/13

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The Sweet Smell of.....E.coli?

Today we retrieved the PCR products part 120 amplified with old primers that were designed by Jared, Diveena, and Aaron. Instead of directly placing these on a Gel and analyzing, we decided to go ahead and double digest PCR products 120 before analyzing. This wouldn't effect the expected band size and if we did get positive results we could go ahead and plan out are ligation process. After double digesting part 120(amplified with new primers) with Xbai1 and PST1 and double digesting the part 120(amplified with old primers) with xbai1 and spe1, we loaded the samples on the gel. The reason for using two different enzymes in these digests is due to the primers that amplified them. The old primer was designed with a longer bp length and the Pst1 site was amplified out, this would make it non-biobrick compatible. The new primers were designed shorter incorporating all the required digestion sites making it biobrick compatible. However, these primers have a higher tendancy to form primer dimers making it less efficient. This is why we ran the PCR for this part at a lower temp. The Gel results contained all the predictions made based on the part sizes and the plasmid size of 119. So, we are also going to go ahead and purify the part 120 with all of the proper restriction sites getting it ready for ligation into the UV promoter plasmid 5001 double digeseted and purified on 11/11/08.


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