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Some students' questions about the lab, and my answers.
Q:"I can't sift through all the cryptic lab background information to find a means of determining DNA concentration."
A:If you mean calculating the concentration of the DNA that we isolated in the second part of the lab, it is the same calculation you did on the take home exam question #1, using the number from the spectrophotometer that we measured last Friday.
If you mean the amount of DNA that we transformed, info you need to calculate the transformation efficiency, just use the same number used in the example in the lab handout (that shows how to calculate transf. eff)
Q:"I can't find a means of locating restriction sites on the vector."
A:I am not sure I entirely understand your question but I will make a stab at it. The only sites on the vector that you know about are theEcoR1 site and the PvuII I site. The vector was cut open (linearized) using EcoR1, and the KanR insert with EcoR1 ends was ligated into that. The PvuII site lies outside of the insert in the newly created plasmid. The insert contains a ClaI site.
Q:"Figure 3 refers to vectors and vectors while the body of the lab text talks about vectors and inserts"
A:The body of the lab text keeps it simple and only discusses the most likely scenario, and the situation you are aiming for--one insert in one vector. The Figure is trying to discuss all the possible ligation products. because the vector and the insert both have EcoR1 sticky ends, they can stick to together in many possible ways. Vector with vector. insert with insert. Two vectors and an insert etc. Some of these weird ones are eliminated during process of transformation, because large and or linear fragments do not transform well. Some are eliminated after transformation by the selective media--anything without an insert will not grow, things with only insert cannot replicate, so only things with vector and insert will grow on the plates. The restriction digestion is done in part to figure out what particular recombinant vector you created--it will be some combination of at least one insert and at least one vector.
When you do your digests, each one will tell you something. Cut with EcoR1 (lane 3) and ideally (if you have a "normal" one insert plus one vector situation) you get a band of 1300 (insert) and 3000 (vector). It should look the same as DNA in lane 7. If you get somethings else, it is your first clue that something weird happened. Digestion with PvuII should result in a single band of 4300. The PvuII ClaI digest is meant to determine the orientation of the insert in a one insert one vector situation. If you got something strange, this digest may help to figure out what is going on. I find that drawing some possibilities with sites drawn in can help figure things out. There is a discussion about possibilities on pages 11 and 12 of the part 2 handout.
Q:"There's a whole lot more I'm not understanding, but I guess I can't ask for all the answers."
A: Sure you can--the lab reports are supposed to be a learning experience not a figure it all out on your own while beating your head against the wall experience:) Ask away...
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