QUESTIONS THAT CAME UP

1) HOW MUCH INFO FOR FULL MARKS?
It depends. If it’s just a table to fill in with « + » and « -« , then you don’t need to give much info at all.
For restriciton mapping, you will want to show your work (bunches of circles or lines all scribbled with one enzyme or the other…), but make sure that YOU SHOW THE FINAL ANSWER CLEARLY, neatly, and with all the symbols that you invented properly explained/defined.
For experimental procedures, you’ll have to give alll the key info without unimportant details. To identify key info think about things like would the strain of bacteria that i use matter? Does it have to be a specific genotype? If so, indicate it, it’s key info! Would I need to plate on a specific medium? Where would I get mutants from? What is the source of the DNA that i am proposing to clone? All key info! What would I be looking for in my screen?

Some technical info on the various types of blot, on PCR, etc, can be found under the link "techniques ch11 and 12 stuff".




2) HOW TO DO RESTRICTION MAPPING?
First, it’s important to realize that restriction enzymes simply recognize specific sequences and cut the DNA within those sequences (they are like cissors).
Restriction mapping is an ANALYTICAL TECHNIQUE. You always do complete digestions for restriction mapping (unless the enzyme does not work properly and the reaction ends up being incomplete).
In order to determine a restriction map, you need to know the sizes of the restriction fragment produced by your enzyme(s) of interest from your initial DNA molecule. You do this by running the products of the restriction digestion on a gel. You also need to know if the initial DNA molecule is circular or linear.

Short explanation : for each restriction digest, draw a molecule (circle or line) and place restriction sites for the enzyme used. Then look at double digests and try to superimpose the maps for the two corresponding single digests. And so on. It’s a lot of trial and error.

Long explanation :
To analyze the results of a set of restriction digests you start by drawing out your DNA molecule (a line or a circle, depending on the situation) and look at the products of one single restriction digest, let’s say with enzyme RA. If you see one band on your gel, and it is 12kb, and your DNA is circular, then it means that enzyme RA likely has only one site that it recognizes. You can draw it anywhere on your circle.
Then look at the results with enzyme RB, let’s say you see 2 bands, one that is 4kb and one that is 8 kb. It means that RB cuts at 2 sites that are 4kb away from each other. You can draw that on a new circle.
Now you’ll probably find the results of a double digest with RA and RB. Let’s say it gives you 3 bands that are 5kb, 4kb and 3kb, respectively. Look for a band size that is in common with either the single RA digest or the single RB digest…here we have the 4kb one. This suggests that the bands of 3 and 5kb represent a ‘chopped up’ version of the 8kb band that you had in the RB digest. And what could be chopping it up? Probably enzyme RA cuts somewhere within that 8kb region, at a site that is 5kb away from one RB site and 3kb away from the other.