Double digestion and ligation problem - (May/17/2004 )

I want to insert my fragment into AAV vector(6.1k). I try many ways for many times, but failed. So I try to do some control test to find the problem:
(I want to find if both RE work well)

I used to use two RE together: BamH1, EcoR1 ; Xho1, EcoR1 for another fragment.
This time first I use BamH1 alone (Xhol 1 alone in anthor group), then test by gel, the plasmid is cut, I can see a band at 6.1k.

Then, I use some of these cut vector (purified) and digested by EcoR 1. (also a band at 6.1k, so i am not sure both RE work by now)

Now I have three kinds of digested vector: digested by only one RE, digested by two seperately(step by step) and digested by two RE together(as before). (actually, I have six groups not three: BamH1/EcoR1 and Xol1/EcoR1 double the number)

I try to ligase all these cut vectors with T4 ligase, I want to see the group digested by only one RE can be ligased and will have a same band in gel as the vector have not been cut, if the vectors are cut by two different RE, ligase will not occur or is not as easy as the vector cut by only one RE. So I can make sure the digestion is successful or not.

But the result is completely not as I supposed.

All bands in every group are above 9k(complete circle vector has three bands at 4k(main), 7k,and 9k), and is not exactly the same position with each other. They are not seem to be ligase as a two-vector-together, at lease there should be some self-religased vector, right?

Or this means there is some problem with the ligation step: the ligase or some other factor? or self-religased vector will change the shape so will not as the same as the vector never been cut?

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Sorry, i'm not sure i understand everything......

but maybe some helpfull hints:

Your uncut plasmid has 3 bands > suppercoiled DNA runs fastest (the 4k band? this should be the main band)
nicked DNA (single strand break) will release the supper coiled state. THis runs the slowest (the 9k band)
linear DNA (double strand break) will run at its expected size (7k band)

Now all this lgaion stuff will be quite dificult to interprete.
Only if the unligated fragments look different from the ligated ones then the ligase probably works.

You should get a proper sequence map of the vector (and insert fragment/vector)

Then do some test digests to see if the fragments you get make sence. So use multiple cutting enzymes or double digests.

Then if you are concerned about two sites (eg BamH1 EcoR1 and Xho1) do single and double digest with those enzymes and another enzyme that will give a fragment of a given size (eg 1kb and 5 kb).
This will tell you that they are all present and the vector is Correct.

Then digest your vector with the two enzymes you need to use say 3-4ug in 150ul digest for 1.5h 10overdigetion so 3ul enzyme of each, add 2u CIP(phosphatase) for 30' and run it in a rather wide slot on a say, 0.7% gel.

Do the same for your insert but no CIP (run on apropriate gel).

Check 2ul of each on gel to make sure there is inded DNA.
setup the ligation 1ul vector 3ul insert upto 10ul total.
Ligate O/N at 16C
transform the most competent bacteria you have and do a collonie hyb if you need to.

You need only one clone!

good luck.

PS. Just make sure you check your enzymes in the NEB cataloug. Be careful with BamH1 i think it has star activety in some bufferes.

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You cannot see ligase activity on a gel. Too many types of reactions occur generating smears

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Thank you for your suggestion.

Now I have inserted one of my interested DNA into the AAV vector, I think the problem before is digestion. The reason is (I think it is intersting):

As I did,I digest the vector by some different ways, and try to religase them by T4 ligase. The result is observed on the gel electrophorisis:

1. an complete (never cut) vector: three bands: 4k(main), 7k, 9k.
2. all cut vector(by only one RE or two): one band at 6.1k

both above results are ok

3. re-ligase vector:
a/ digested by only one RE: a band above 9k
b/ digested by two RE together: two bands: one is the same with the band(above 9k) of a/(digested by only one RE); the other band is above the first band
c/ digested by two RE but one by one: two bands, the positions are the same as b/

all bands are above 9k but not the same with the never cut vector, maybe the shapes of the vector are changed after ligase. The lower band maybe the re-ligased vector, but what is the upper band? (occur in try to ligase a vector digested by two different RE)

So I do somthing then:
Re-Digest with one RE(the same one in tests above):
And I get two bands.(why?)

I get the band at 6.1k again: it is the cut vector; and another band is about 7k in a/, b/, c/ all three groups.

I really do not know what the upper band is.
the band at 6.1k in the group digested by two RE one by one is slight, so it may mean religse is slight , and the digestion by two RE is more completely, so I choose the vector in this group for further use, and get my DNA inserted.

But what about the other band?

I donot know if I make myself clear,sorry.

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It sounds like you have multiple inserts, which you can find out by doing restriction mapping. When checking if you have what you wanted I always choose a RE that is unique within the vector and another RE that is unique within the insert. If your total size is different you might want to try to grow your bacteria after transformation at 30C.

Good luck!

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Hi guys:

I am cloning a PCR product as 350 bp into pCDNA3-1. I have some troubles with first cloning. After screening 20 clonies I just got 1 clone which included my insert. I went to determin the orientation of insert. So I used EcoR I and HpaI. Both have 100 pecent activity in NEBuffer 4. But after digestion I got two bands one related to my plasmid and second one was bigger as almost 600 nt? I am thinking about the contamination of my enzyme, for I already tested vector with both enzymes individually. Would you please let me have your comments? I appreciate your help.

All the best

Haki

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What the molecular size of pcDNA3.1 after double digestion? Is it still about 5.4 KB or less? If it is less than 5.4 kb, mayebe the reasone is the contamination of your enzyme or star-activity of the enzyme(added more enzyme or used improper buffer). You can show your gel picture here.

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Hello

I have a concern about DNA ligations.

I am trying to ligate 2 DNA fragments cut with kpn1 HindIII (1.3kb)and the other with KpnI and XhoI (6.3kb). I am only getting intramolecular ligations. I need to insert the ligated 7.4kb DNA in a vector cut with Hind III and XhoI and then clone it in E coli. I am using T4 DNA Ligase. Also its very difficult to detect the DNA in the gel as the quantity is too loo. What should I do about it.
Any suggestion will really be appreciated.
Thanks

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first of all if you are trying to check if the digestion is working properly or not, why are you going for a ligation step.
what are the problems you faced during the cloning earlier, please send those details so that one can try to pin point the prblem there itself.

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check if there is any kind of contamination in your cells in wich you are transforming your plasmid.

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