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A Flower of A Different Color
Sushma and Binu worked on the extraction of plasmids (from the overnight cultures), containing our selected promoters. We used the kit, GeneJet for the miniprep and stored the DNA, suspended in elution buffer, at -20 degree celsius. We also made glycerol stocks of the same plasmids.
Melissa, Rajiv and Tiffany worked on RNA extraction for phalanopsis flowers using the Rnasy RNA mini kit. To prevent overly heating the product before RNA extraction we ground the tissues in the cold room. And flash freeze the tube and centrifuge while we were weighing out the product. We also completed PCR on the DNA extracted from last lab, testing different quantities of DNA 1mirco liters, 2 micro liters, 4 micro liters, 8 micro liters and 10 micro liters in two temperature ranges 53.7C and 58C. The PCR products will be further tested with electrophoresis. If the product does appear to have bands in the correct range then the product will be offically sent off to Iowa State for sequencing.
Angie & Diwash performed a restriction digest of extracted plasmids pSB1A3 & pSB1A7, which were extracted by Axel using the Gene Jet Miniprep protocol last week. These plasmids were extracted from overnight cultures of glycerol stocks of E. coli from MIT containing these plasmids. We used fast-digest enzymes, SpeI & XbaI to digest the plasmid DNA,then ran electrophoresis of the plasmid DNA for 40 minutes at 150V. We obtained much better results with this gel than previous gels, which is probably due to the change of protocol for the extraction procedure. The new protocol Axel used seems to produce much cleaner, purer plasmid samples. The bands of the plasmid DNA were visible for both plasmids, but the number of base pairs expected was not correct. pSB1A3 should show a band at 2157 b.p and pSB1A7 at 2431 b.p, but we saw the opposite in our gel. We saw a band around 2,000 b.p for 1A7 and a band around 2,500 b.p for 1A3. It should be the other way around. It is possible that the plasmid samples somehow were switched or mislabeled in our research, or maybe even the MIT glycerol stocks containing each plasmid could've been mislabeled before being sent to us. Some of our previous gels show bands around 2,000 b.p for 1A3, like we would expect, but most of our samples of 1A7 have not looked to of been properly digested, because many times, bands don't even show up in our gels. Maybe the extraction procedure we have been using previously (GET/Miniprep) produced 1A7 samples which were too unpure, dirty to get good results in our gels. Since we don't know for sure if the plasmid samples we are using were accidentally switched, we are going to check the identity of these 2 plasmids by performing PCR on Thursday. We will use 2 primers we have available to check the length of a known segment which shows up in both plamids, but differs in length in each. We can then move forward with our vector work.