[nelson]

Hi,

We did an experiment to produce a restriction map from a single digest of EcoRI and HindIII and a double digest of both these on pUC18 with an unknown foreign insert. We ran through a gel and Ive attached the image.

Ive done a lot of research into this but still cant work it out. There is 1 cut site for each of these enzymes on pUC18 one at either end of the MSC, but in HindIII lane i only get the whole plasmid and in the double digest I get the Plasmid plus 2 fragments.

For one all the fragments do not add up to make an explainable total and why is there no fragment in the hindIII lane but there is in the double digest lane?

Hope somebody can help.

Thanks
Neil.

[Grundalizer]

Can you please give us what is in each of the numbered lanes?  I've done some work with both those enzymes, but need to know what you put in each well.

[nelson]

oh sorry i did lable the diagram but not in the post....

Lane 1 - Bioline Hyperladder 1
Lane 2 - Uncut Plasmid
Lane 3 - HindIII
Lane 4 - EcoRI
Lane 5 - EcoRI + HindIII double digest

Again my apologies.

Thank you for the reply.

[Grundalizer]

I slowly strayed away from micro as I got into college, as I'm a chemistry major now, but did a summer internship where I used exclusively HindIII and EcoRI.  That being said...I'm still trying to figure it out.

I did find someone saying this, "The difference between pUC18 and pUC19 is that the MCS is reversed. On pUC18, Hind III cuts at bp 399, and Eco RI cuts at bp 451, liberating a 51 bp fragment and a 2,686 - 51 = 2,635 bp fragment." on a protocol site...even though that probably doesn't apply here.

Would you mind sharing the size of the fragments?  I also find it strange HindIII doesn't show that second smaller fragment, but the double digest does.  How small are the second frags? 

Alright...let's try and figure this out, I've recruited some of my bio major friends to look at this also.

[Grundalizer]

Did you use the same HindIII enzyme stock for the lane 3 digestion and lane 5 digestion?  EcoRI's secondary fragment shows up fine in Lane 4, and again in Lane 5, so EcoRI is working fine.  The fact the second small fragment shows up in Lane 5...I'm guessing is where we should also see a band in Lane 3, as it is caused by HindIII.  If you used the same enzyme stock (which I assume you did) I find it strange that secondary band doesn't show up in lane 3.  I also don't get why Lane 2 is higher than Lane 3.

In the double digest..we see what should be the length of the MCS, since it should theoretically be cut on both sides, since HindIII and EcoRI are on the edges of the MCS...I'm not seeing why there would be more than 1 fragment though

[Grundalizer]

You said you've done a lot of research into this...but have you run more than 1 gel?  Is this a theoretical question posed by a professor or something or is this a gel you ran and photographed?


NOTE: My bio major friend just noticed something.  There should NOT be the secondary line in Lane 4, as only EcoRI should be making the single cut...leaving just the plasmid.

He also said there should only be 2 lines in lane 5, the MCS fragment and the cut plasmid.  Was there contamination?

[Grundalizer]

Ok, talked to my friend for a while again.  He said that when these 2 enzymes are used together, there is star activity on EcoRI, which means there is unintended cleaving.

Check here.

http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

Put in those 2 enzymes, and you do see star activity on EcoRI, which explains the double digest...also, it MIGHT explain why in the EcoRI lane there is another unintended fragment, possibly due to a little HindIII contamination.

He also ads, partly in question...did you have an alkaline phosphatase in there?  Because if that's not in there to remove the 5' phosphate, the plasmid can close right back up.

[Wreath]

Well, since i found this
http://www.bio.utk.edu/peterson/BCMB%20515/Vector%20sequences.htm#I.RESTRICTION%20SITES
it seems to me that you have some contamination.
These fragments on 4,5 are about 270 BP, the new single lets say 350 BP. But they cut only in single place, so they only (on its own) should linearize the vector, making one band. That band in 3, size around 3000, could be the Hind III digest. Let's say that the band line 2 really is your plasmid, although it's about 2000 now (can be because of it's circular form, I'm not very optimistic about the 1000 BP marker difference only by circular x linear form, but let it) and I have not a clue about the large bands (unclean isolation?)
but what does not make any sense to me are lines 4 and 5, EcoRI should make the same band as HindIII. The double digest problem I believe can be because of star activity as Grundalizer posted.
I would like to know, what was the load you put on gel and how did you get the plasmid (buy x isolation). In fact, the greatest thing you could do to help us help you is put here and entire protocol :-)

[nelson]

Hi guys thanks for all your replies.

The sizes I work out the smaller fragments to be are -

EcoRI - 300
Double Digest - 300 and 400

My initial thoughts were that there had been some contamination or HindIII hadn't correctly cut, But my professor said that the gel was pretty much perfect and I should be able to work the restriction map out from it.

I'm not sure about alkaline phosphatase though, we didn't add any ourselves but there may have already been some added.

I will post the protocol once i can get my scanner working.

Thanks again.
Neil.