A FEW ANSWERS

I am sensing a lot of midterm-related stress...
Here are a few things:

1) no more practice questions in my mailbox, and my originals are gone too! However...your colleague Belinda has them all scanned, I think, and offered to send them out to her classmates (thanks a million!). Her email address is in one of the comments-and we all owe her coffee!

2) I am looking at a double digest question from the 2006 midterm (page 4) that is online. I will do it and report each step. I hope it helps.
- circular molecule
- SstI + SapI give 2 bands--> there probably is only one Sst and one Sap sites, they are 0.5Kb apart, the total size is 4.3 (matches with info given)
-Sst+ BglII--> 2 bands, so only one BglII site, 0.6 kb away from Sst. At this point BglII could be either 0.1 away from Sap or 1.1 away from Sap
- same idea for Sst and Xho, they are 0.9 kb apart
- we are told that cloning into (i.e. cutting with) Sap or Xho inactivates the gene for kan resistance, so both Sap and Xho must be within the kan resistance gene. Since cloning into (i.e. cutting with) Sst does not break the kan resistance gene, Xho and Sap must be 'on the same' side of Sst; thereofre Xho is 0.9-0.5=0.4 kb away from Sap. Also, BglII does not fall into the kan gene (does not say that cloning into it breaks the kan resistance gene) so, relative to Sap, it must be on the opposite side of Sst. So, BglII is actually 1.1 kb away from Sap
- Hpa is 1.1 away from Sst, but we don't know on which side. Hpa is also 0.5 away from BglII...now: if Hpa was on the same side of Sst as the kan resistance gene, then it would not be 0.5 away from BglII! (Are you still with me? Try drawing it out...). Therefore HpaI must be on the same side of Sst and BglII, just a little further (0.5 kb further away).
-Finally, the Nco site must be placed so that it is 2kb from Sst and 0.9kb from Hpa
The kan resistance gene covers Xho and sap, but none of the other sites

DONE!

3) It's very rare that you have 2 restriction sites at the same location on the chromosome at the base pair level...However they may be very close and cut 3 or 4 bp apart...in such cases we won't be able to tell the size difference.

4) extra help: I'll be in the help desk tomorrow, friday from 12.30 to 1.30 if you have any questions, and Saturday from 4 to 5 pm (I can let you into the building at 4pm).

Keep it up, you are doing well!

Cheers

Pam
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EXTRA HELP SESSION(S)

I'll be in the help office on Thursday from 4 to 6 pm. I could be persuaded to do some extra help sessions on friday morning (I have a workshop 2-6pm) or sometime during the weekend for about 2 hours.

You people decide (deadline: Wed. at 10pm)!

Pam

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.