Puzzle about plamid and its digest, plz help - (Feb/27/2012 )

Hello guys, I am new to this website and I must say the discussion topic here is excellent.

I am puzzled by two questions asked by my professor, i tried a lot and was at dead end. Hope u guys can help me.

1) How can you tell if freshly prepared plasmid DNA treated with HindIII has been cut or not?
2) If I give you two unlabelled tubes, each containing a different plasmid of known sequence and size, how could you easily figure out which one is which?

Hope u guys will reply soon.
P.S. in both these questions I am looking for easiest method (I don't know if there is any method apart from electrophoresis or not)
THANK YOU

Hello,

I have no experience at all but I think I might be able to help. However, you might try and check with someone else.
So, regarding the first question if you know the sequence of your plasdmid and the HindIII restriction sites you can predit the sizes of the bands formed and run a agarose gel.

The same applies to the second question, if you have the sequence of the plasmids and their size you can find out their restriction sites, use some uL to do the restriction digestion and run a gel. Because you have their predicted sizes and number of bands (not all plasmids have the same number of restriction sites for all enzymes) you can easily find out what's what.

Good luck

The above answers are only half correct, however, seeing as this is obviously homework, can you tell us what you have figured out for yourself, so that we can help you learn rather than just supplying the answers?

bob1 on Mon Feb 27 20:33:47 2012 said:

Thank you,
You are right, these are home work questions. In the first question, I first thought of using simulation software to predict the restriction site and then to run the electrophoresis, but the question does not mention that the sequence or identity of the plasmid.So, it was a dead end for me since, the plasmid is not known and I have to find that whether it is digested by HindIII or not?
In second question also, I am thinking about electrophoresis.
BUT, I guess in both these questions, the professor is asking about the easier method than electrophoresis (as it is time consuming). I am not sure, but i guess he wants answer not related to electrophoresis. I was wandering, is there any visual or spectroscopical method (using A260 may be) to easily determine whether the plasmid is digested, or to determine identity of unlabelled tube of plasmid. The lack of experience is restricting me to think beyond electrophoresis. Please
P.S. The question I wrote are in exact words that professor gave in written.

Rute on Mon Feb 27 14:27:57 2012 said:

Thank you
I thought of the same thing. But, in first question it is not mentioned that the sequence or size of the plasmid is known. While in the second question, we can do digest the plasmid and do electrophoresis but, I guess, the professor is expecting easier method for identification (Electrophoresis would take about 1 and half hour). What I am looking for is that is there any visual or any another method to determine whether the plasmid has been cut or to determine the identity of unlabelled tube of plamid.

Ok, you are heading off on a tangent. Electrophoresis isn't terribly slow, I can run a plasmid gel in less than 10 minutes from loading to visualisation, with the right buffer.
For question 1 - what do you know about the forms of a plasmid (hint - there are 3, and are independent of the sequence)?
For question 2 Darius was correct - you can predict the digestion fragments sizes if you know the sequence and the plasmid size. It will help if you also know what form the majority of the plasmid is in.

bob1 on Mon Feb 27 21:06:57 2012 said:

Thanks,
As far as i know there are three forms: Nicked, linear and supercoiled (covalently closed circular). I think I am getting somewhat of what you want to say. You want me to perform electrophoresis and to determine whether the plasmid (digested by HindIII) is in linear state or whether it shows more bands on gel. If it is found to be linear or shows more bands then it can be inferred that the plamid has been cut by the said enzyme. Please tell me if i m wrong on this conclusion.
About the second question, I guess u are saying that I should use the simulation software (NEBcutter) to obtain virtual digest of the plasmid using different enzymes (since, contradictory to question 1, in this question 2 we know the size and the sequence of the plasmid); and then you want me to confirm the identity of the plasmid by running the gel and comparing the results with the virtual digests.
If I am wrong on both these assumption, please do correct me....

Yes, correct on both counts. The three forms are as you said, and you should see each form on a gel - if you digest with HindIII, these will disappear and be replaced by one or more bands that will be either linear plasmid (in the case of a single cut) or fragments from a multiple cut. If you run these next to plasmid that you know is uncut, you can compare.

You can also do the comparison digest manually if you need to rather than doing it by computer. Note that any small fragments get progressively hard to see on a gel, as they only make up a small fraction of the total DNA loaded and the smaller they are the smaller the amount of ethidium bromide they will intercalate so it makes it more difficult to visualise. You may need to load several micrograms if some of the fragments are less than 500 bp.

bob1 on Mon Feb 27 23:23:45 2012 said:

Thank you very much sir,
I will definately tell you , what my professor has to say about the answers of these question (i.e. he will tell answers after correcting the paper- may be after 15 days).
Thank you once again for your suppot.I do have lot of questions but I guess they will get somewhat clear after performing the experiments.
I guess you would have realized that the questions I posted where the part of the post lab home work.