DNA plasmids and restriction enzymes?

Question

When running an agarose gel electrophoresis of a DNA plasmid with different restriction enzymes how do you determine the molecular weights of the fragments generated from each of the digest reactions?

I'm trying to finish my lab report (~1/3 done) and still be able to get some sleep before my lab tomorrow morning....thank you for your help.

Answer 1

I believe the following links reiterate Odyssey’s answer: http://micro.magnet.fsu.edu/electromag/java/electrophoresis/
http://www.rit.edu/~pac8612/electro/Electro_Def.html

Hope this helps

Answer 2

you ca use a restriction map of the plasmid that indicates exactly where the restriction enzyme will cut the plasmid.

The map normally shows the molecular weight.

or just use a molecular weight marker in the electrophoresis gel that migrates according to the weight. You then compare the migration distance of the weight marker to that of the fragment that you seperated.

Answer 3

Blunt end cloning is rough. There are certainly ways to do this that I"m not aware of, here's how I would approach it (7 years molecular bio experience) 1. Restrict the plasmid. 2. Dephosphorylate the plasmid so that it cannot self-ligate. Clean. 3. Ligate in your oligo (make sure that it IS phosphorylated). Blunt end ligation is very inefficient, so let it go for a long time. 4. Transform into new strain. 5. In theory, if everything has worked as planned, you'll only have two possible clones: those plasmids with the insert in one direction or another. In practice, you will observe plasmid autoligation in addition to other less easy to explain anomolies. You will have to screen a number of transformants. Make a small library for this purpose. 6. Screen. Now, you can go about this in many ways. In this situation, you're probably stuck with PCR screening, either with general primers and characterization of the clones through sequencing OR you can make a primer set that will specifically amplify the correct clone and go from there. Note, because blunt end ligation is so inefficient, make sure you go into this with TONS of plasmid and TONS of insert. Regarding the ligation, go for a 10 to 1 molar ratio of insert to vector. If this doesnt work, its likely that it would work if you worked with more concentrated DNA. Its basicallly a crap-shoot approach: the more shots, the more the chance of success. edit: 1. thanks for wasting my time and then giving me a thumbs down. 2. the answer is either absurdly obvious or you don't understand the question.