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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posted by pam39 @ 8:46 PM
17 Comments:
Where is the extra help going to be held? in your office? or...at biosci genetics help room? Thanks
Hey you guys,
For the 2006 practice exam, #2 on pg 3. for part A, how can you tell which one is the reading frame? Did you just find a distance b/w a start and stop condon that is 360bp long?
Thanks
Help desk in room 2519, biosci building (Craig's office, usual help desk room)!
OMG!!! This midterm is going to be horrible
I think we're all so frustrated because all of our practice exams do not have answers. and discussing answers online can be so limited =(
about the ORF (3600bp): yes!!
Please do not panic. Many of the practice questions come from midterms that were later on in the year and therefore need a little more background and practice than what you have right now. You'll be fine!
Cheers
Pam
Hello, I have a problem on positive regulation. From the lecture notes, when the repressor binds to operon no transcription occurs. If the repressor is not binding to the operator while the CAP is also not binding to the promoter, it seems that there will be a small amount of transcription. Is my understanding correct? Does this small amount of transcription enough for the E coli to survive?
yes that is correct! when you have positive regulation there will always be some transcription occuring even if the CAP is not bound, but the transcription is amplified when CAP does eventually bind. This is opposite of positive regulation, when there is no binding of a repressor protein, there will be extreme transcription...LOTS...but when the repressor does eventually bind, there will be a cease in transcription all together.
Hope that helps
Just a question continuing on with the CAP stuff. So when there's a mutation in the crp gene and it is Lac I (s) does it mean that there is still going to be low levels of transcription b/c the CAP is a positive regulation? or it is that nothing will be made because of the super repressor?
thanks
Can we assume that whenever we have a mutation in the crp gene in the genotype but a proper crp gene in the plasmid that transcrpition will never reach its full potential?
Hi Pam,
I'm just a little confused between the differences b/w catabolic and biosynthetic operons. So is the major difference between the 2 is just WHAT they bind to before they "induce" negative regulation?
How would they test us on the exam about this?
Thanks for your help!
Question:
I know that the role of PCR is to amplify the gene that you want in lots and lots of fragments but, under what circumstances would be do this? Like for example in exam questions...what information in the question could tell that we would need to run a PCR?
Thanks for your time.
i think in order to run a PCR, you need to be sure that you have the sequence of the clone in order to choose the primers needed for PCR. mmm i'm not sure though but you have to be pretty sure that you have a full map of that clone so you know extractly the lcoation for the primers.
Re: Can we assume that whenever we have a mutation in the crp gene in the genotype but a proper crp gene in the plasmid that transcrpition will never reach its full potential?
mmm i think the crp gene works in trans in the system. so if the chromosomal crp gene is mutated (like it won't make any CAP) but the plasmid crp gene is not then the CAP from this gene will work in trans to the chromosomal gene. Right? similar to the mutation in lacI- and lacI+ or lacIs....i hope this makes sense....and if i'm wrong please correct me....
good luck studying :)
Re: Just a question continuing on with the CAP stuff. So when there's a mutation in the crp gene and it is Lac I (s) does it mean that there is still going to be low levels of transcription b/c the CAP is a positive regulation? or it is that nothing will be made because of the super repressor?
since there is a mutation in the crp gene then no CAP is being made....and plus if there is a lacIs then the mutated repressor will block transcription....as a result, there shouldn't be any transcription occurring....
:)
Thank you :) person
I see some great discussions going on here!
The whole crp mutant/CAP/lacIs/positive regulation that someone explained above is absolutely correct.
[The way I think about it is: binding (or not) of the lac repressor to the lacO determines whether there will be expression (yes or no). Binding of the CAP will determine, in case there is expression, whether there will be a lotof expression or only a little. The binding of the CAP protein is irrelevant if the repressor is bound to lacO].
For the PCR, the 'trick' is that when we build a clone (or a whole genomic library) we use a plasmid (vector) that we know the sequence of. So, we can easily design primers that are within the plasmid sequence ('bordering' our unknown insterted DNA).
All the best!
Pam
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