Lab 8: Restriction Enzyme Digestion of Plasmid DNA Flashcards
Restriction Enzymes
enzymes derived from prokaryotic organisms that were originally meant to degrade foreign phage DNA that has been inserted into the main chromosome by cutting it out as a form of bacterial self defence
Plasmid:
an autonomously replicating DNA molecule that is not found integrated into the main bacterial chromosome. it is useful because it often aids in the bacteria having antibiotic resistance.
Why are plasmids useful in genetics?
In genetics, plasmids are useful for DNA amplification because a desired sequence can be inserted into a plasmid and get replicated, then you can cut it out.
Recombinant vs non-recombinant plasmids
recombinant plasmids have forein DNA inserted into the original ring, and when they undergo an RE digest, they show 2 bands in the gel after electrophoresis.
Non-recombinant plasmid only contains its original genome dna, and after undergoing an RE digest, it still leaves only one band.
Uses of tracking dye
tracking dye is added to the DNA solution in order to stop the restriction enzyme reaction from continuing to cut up the plasmid. It also aids as a visiual of electrophoresis completion, so you dont run your dna off the plate. It also helps with providing “weight” to the sample, in order to displace buffer in the wells. it does not stain dna. Only SYBR safe does that
What does SYBR safe do?
DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels. it is in the gel, and it binds to DNA as it runs though. the gel can be exposed to uv and you can see where the DNA stopped because of the SYBR safe.
Are large chunks of dna at the top of the plate or closer to the well.
the large fragments will not move as far away from the well as smaller fragments, which can migrate further.
What kind of RE and plasmid were used in this lab?
Used HINDIII, used Plasmid YCplac33-5600bp. 4 plasmids were used, one was the original YCplac33 plasmid, the others were recombinant, with foreign DNA inserted at the MCS gene spot near the HINDIII REsite.
The plasmids got digested with HINDIII and trackerdye was inserted to stop reaction. gel was ran. it is expected that the hindIII will cut the forein DNA in the recombinant plasmid out because it is attached to the RE sites, therefore, recombinant plasmids will show two bars on the gel.
