Plasmids restriction enzyme question

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Plasmids restriction enzyme question

Post by georgina_009 on Wed May 07, 2008 6:52 am

Sorry to overload you with qns but this is another thing I cant understand:

Plasmid pUNE92 has been digested with BamHI and HindIII restriction enzymes in various combos. the following DNA fragments sizes were seen:
BamHI - 4.0 + 2.0 kb
HindIII- 6.0 kb
BamHI + HindIII - 3.0, 2.0 1.0 kb

a. What is size of the plasmid? my answer - 6kb
b. How many sites are cut by each enzyme? - my answer - Bam - 2, Hin - 1, Bam Hind - 3?
c. This part Im not sure how to do - Draw a restriction map, indicate the position of the sites in terms of hours on a clock face. Could you help me understand how to do this and tell me if my answers are remotely correct?

Thanks alot for your valuable help!

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Re: Plasmids restriction enzyme question

Post by Alla on Thu May 08, 2008 3:03 am

Your answers a and b are correct. As for the other question:

You have 2 enzymes. You know the sizes produced by each one. Now you need to examine what happens to those sizes when you use both to find relative position of each cut.

Obviously since HindIII cuts only once you need to examine the sizes of fragments produced by the other enzyme. So BamHI alone produces 4 and 2 kb fragments but in the digestion with 2 enzymes it produces 3, 2, and 1 kb fragments which means that 2 kb fragment remains untouched and 4 kb fragment is digested by HindIII into 3 and 1 kb.

Your plasmid than is (join into the circle):

---BamHI ----(2kb) ---- BamHI -----(3kb) ---- HindIII -----(1kb) ---

All you have to do is draw a circle and draw restriction sites in the proper positions (easily done since 1 kb would be equal to 1 min in this case).

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Re: Plasmids restriction enzyme question

Post by georgina_009 on Thu May 08, 2008 7:12 am

Ok but how would you do one with loads of sites?
I have another harder one that has
PVU - 2.2 0.3 kb
Bam - 2.6
Sca - 2.6
pvu/bam - 2.0 0.6
bam/sca - 1.6 0.72
pvu/sca - 1.5 0.66 0.3

The numbers don't seem to add up if the whole plasmid is 2.6

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Re: Plasmids restriction enzyme question

Post by Alla on Fri May 09, 2008 12:11 am

You are right, this problem does not make any sense. The Pvu/Bam restriction should not produce 2.0 and 0.6 kb if Pvu alone produces 2.2 and 0.3 kb (unless partial digestion involved). The numbers that donít add up pose less of a problem since any fragments that are too small will not show up. Please, make sure that you copied the problem correctly and did not leave out any instructions (also, was the vector size given or you assumed what it was).

Please, let me know. I will do my best to help you with this.

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Re: Plasmids restriction enzyme question

Post by georgina_009 on Fri May 09, 2008 5:32 am

I had to measure them out and find the sizes myself from a gel photo. I must have done something wrong! I'll have to start over. Thanks anyway

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Re: Plasmids restriction enzyme question

Post by Alla on Tue May 13, 2008 4:19 am

You are welcome.

P.S. It might be easier if you posted photos for problems like these.

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