A few answers
I see that there is some action going on here, nice job everyone!
Let's try to address your concerns in some sort of organized way:
-I don't think Omid's mock exam is available yet, I tried to download it too and it wasn't accessible.
- your midterm predicitons seem pretty sensible to me: lac operon-type (prokaryotic gene regulation) stuff, restriction mapping and a possible cloning project....sounds good! But please realize that I don't know what Craig is planning on doing.
Cloning project: we've seen, so far, 3 basic approaches (functional complementation-based cloning, cloning based on sequence homology, and you guys did cloning based on differential expression/expression profiles in class). Note that not all these approaches need a probe! The first one, for example, is based on restoring a function, and you don't need any probes to check for a function.
About the outlined cloning procedure:
-first of all, you need to know what type of gene you are trying to clone, and what information is available!
- you need to say where your DNA, that you partially digest, comes from (a bacterium? plant? fungus? Is it genomic DNA? Is it subcloned DNA? etc)
-you ALWAYS want a selectable marker in your plasmid, typically a drug resistance. People do not use the LacZ+ as a selectable marker because it complicates the procedure (you can't use standard media, etc). The lacZ gene on the plasmid is used for a different thing...we can go over this later.
-If you are using colony lift hyb. to find the colony that contains our gene of interest, that means you already know the gene sequence....so why wouldn't you skip the whole procedure and just PCR it up from genomic DNA? You need to be very clear about the goal of your project, what you have at yoour disposal and what you don't. Different situations require different procedures!
- you don't run Southern blots on bacterial colonies...you would need 100s of gel lanes, which means dozens of gels!!
NOTE that, in all cases, once you have identified a clone that contains your entire gene, you don't need to do anything to it; you got it. To amplify it you just grow up your bacterial culture that contains the plasmid with your entire gene of interest, the next day you do a plasmid prep from that culture, and there you have tonnes of your gene of interest, conveniently inserted into a plasmid. If you want to cut it out it's really easy because you can use restriction enzymes!
See you in class!
Pam
Let's try to address your concerns in some sort of organized way:
-I don't think Omid's mock exam is available yet, I tried to download it too and it wasn't accessible.
- your midterm predicitons seem pretty sensible to me: lac operon-type (prokaryotic gene regulation) stuff, restriction mapping and a possible cloning project....sounds good! But please realize that I don't know what Craig is planning on doing.
Cloning project: we've seen, so far, 3 basic approaches (functional complementation-based cloning, cloning based on sequence homology, and you guys did cloning based on differential expression/expression profiles in class). Note that not all these approaches need a probe! The first one, for example, is based on restoring a function, and you don't need any probes to check for a function.
About the outlined cloning procedure:
-first of all, you need to know what type of gene you are trying to clone, and what information is available!
- you need to say where your DNA, that you partially digest, comes from (a bacterium? plant? fungus? Is it genomic DNA? Is it subcloned DNA? etc)
-you ALWAYS want a selectable marker in your plasmid, typically a drug resistance. People do not use the LacZ+ as a selectable marker because it complicates the procedure (you can't use standard media, etc). The lacZ gene on the plasmid is used for a different thing...we can go over this later.
-If you are using colony lift hyb. to find the colony that contains our gene of interest, that means you already know the gene sequence....so why wouldn't you skip the whole procedure and just PCR it up from genomic DNA? You need to be very clear about the goal of your project, what you have at yoour disposal and what you don't. Different situations require different procedures!
- you don't run Southern blots on bacterial colonies...you would need 100s of gel lanes, which means dozens of gels!!
NOTE that, in all cases, once you have identified a clone that contains your entire gene, you don't need to do anything to it; you got it. To amplify it you just grow up your bacterial culture that contains the plasmid with your entire gene of interest, the next day you do a plasmid prep from that culture, and there you have tonnes of your gene of interest, conveniently inserted into a plasmid. If you want to cut it out it's really easy because you can use restriction enzymes!
See you in class!
Pam
posted by pam39 at 8:38 AM
16 Comments:
So when we're doing a cloning project, we need to know the type of donor DNA. We need to run gels only if we have a known probe? what is the probe is not known, then do we go for functional complementation?
our genomic library or cDNA library is just a digested portion of the genome or cDNA right?
yes, we need to say where exactly the DNA comes from (e.g. genomic DNA from lys- yeast, or cDNA from 3rd instar drosophila salivary glands grown at 18 degrees, etc).
We can run gels whenever we want to-gels are for separating DNA fragments based on their size. Whenever we want to do such a thing, we run gels.
Probes don't need to be "known"...we just need to have one that, for one reason or another, we know will be helpful. We can know its sequence, or where it hybrizises relative to some "landmarks", etc. We always need to specify what our probe is (e.g. a random piece of DNA from mouse chromosome 2, or gene such and such from Neurospora, etc.)
Always think about:
- what you have, both materially and in a sense of "information"
- what you want to achieve
- what you need to achieve what you want to
There's no set answer; every situation is different!
Cheers
pam
Only restriction maps, Lac stuff and an analysis on the midterm right?
What could the analysis be on?
what do you mean by analysis? Do you mean interpreting results from experiment???
So what do ppl think about midterm?
I thought it was hard, especially #2!!!
OMG...it was a horrible midterm!!! It was nothing like what Craig was setting it up to be!!! Where was the easy restriction map that was supposed to help us all??? Seriously, what was going on in question 2 and question 3, was a bit of a mind boggler. Nothing on that midterm was easy. This midterm was nothing like the practice ones we were given to flex our genetics' muscles with. I feel horribly depressed and I honestly think that I am going to fail!!! This is a horrible way to start a weekend, now I, like all of you, will be wondering about the final until we get it back.
We need to have a chat with Craig, the notes and the material were so easy to follow along, but this midterm was beyond me and I feel like a looser :(
Come on, you have a nice sunny weekend ahead-no depression allowed!
I saw the MT last night and thought: question 1 is extremely easy. Question 3 is easy, although the specific scenario is new to the students-it may create some panic, but they have enough time to get over it and think. Question 2 is, well... in my opinion, inappropriate for this course at this time.
You are all welcome to comment on the MT, but don't let it spoil your weekend!
Cheer up!
Pam
I totally agree with you! Like above comment, I, too, feel depressed and frustrated that I couldn't perform on the exam up to standard!!! Now, we all have to worry about what finals will be like!
I am extremely depressed right now.
Seriously, if we panic on question 2, who could really pay a lot of time on the following question especially when the next question is hard as well under time constrain. I am worried about the fianls, too. It seems that nothing that we have studied so far has anything to do with the lecture materials.
Totally agree!!! I was in a state of shock and panic when I read Q 2 that I was not able to fully understand or comprehend what was going on in Q3. I'm sure it was easy, but under such a high stress level, with beads of sweat dancing on my forehead and the fact that the midterm did not have anything that I was prepared for made Q2 and Q3 like climbing a mountain without shoes or water.
Pam, please make our boices known to Craig, you have more weight and you know him better than we do. This midterm sucked and even though we are having a great sunny day, it feels more like it's pouring!!! I'm just glad to know that there are many others that feel the way I do. A very thoughtful, "we're in the same boat", goes out to the first comment at 2:40...you are not a looser!!! The test just sucked
seriously the tutorials didn't even help with the midterm, that's not right is it
i agree with you 8:27pm! Seriously compared to our practice midterm, Craig's midterm was totally different! I guess it might different since practice midterm was from Dr. Beatty. But our midterm was so different!!!
I think that if Craig was going to be giving us a midterm with questions from left field like that he sho0uld have warned us!!! He could have given us similar questions, it was so hard to determine what he wanted. I think that I just doodled some pictures but had no idea what was goin on!!! I think we should all have a talk about it on Monday!!! The questions, with the exception of #1, were not fair and not expected!!! WHERE WAS THE RESTRICTION MAPPING???
i think that even if Craog's test was going to be different from Dr. Beatty's then he should have told us not to attempt those questions an instead he should've given us extra problems he made up
I have a feeling of dropping this course now and take it in January...
Dr Beatty's exam is hard, but his exam questions seem to be more like the practice questions. Craig didn't really give us his own midterm instead the only sources that we have is just the practice midterm online by other prof. This is...not fair to us.
I totally agree...I don't care if this is post midterm stress and disapointment, but the test was hard and horrible!!! Nothing fair about it and now we all have to suffer for it!!! I really LIKED this class now I am dreading going to class on Monday, there's nothing fun about it anymore!!!!
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