20 replies to this topic

[ixsix]

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?

[il0postino]

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.

Edited by il0postino, 22 May 2004 - 07:11 PM.

[phdconsult]

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

[ixsix]

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.

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.

Edited by ixsix, 15 July 2004 - 08:49 PM.

[phakimpour]

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!

[hmohamma]

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

[doctorphilic]

Hi there,

I have ligated shRNA oligos into a pSIREN-Retro vector for SiRNA experiment. I transformed the vector into E.coli and purified the DNA using Qiagen standard protocol. I digested the plasmid with EcoRI and BamHI (double digestion), which is part of the original vector. But after digesting the DNA in buffer 3, I do not see any bands. Also, originally, i tried 17ul of DNA, 0.5ul of BamHI and EcoRI enzymes and 3ul of buffer 3 at 37 degrees. I tried more than 10 different plasmids with various colonies and no band was found on the gel. I then tried to use 10ul of DNA in 20ul mixture. I also replaced buffer 3 with EcoRI buffer using the NEB protocol suggestions. Also, this time I included dH2O but the enzyme concentration is still 0.5ul (for both enzymes). No bands again. How do I trouble shoot? Is my cloning not good? Or is it the digestion process? Any suggestions will be greatly appreciated. thanks so much!

[phage434]

Do you have any DNA to start with? Can you see a plasmid when you run undigested DNA on a gel? If so, I would recommend then trying each enzyme individually as well as together, and running the results on a gel. Are you running sufficient DNA to see a band?

[doctorphilic]

Do you have any DNA to start with? Can you see a plasmid when you run undigested DNA on a gel? If so, I would recommend then trying each enzyme individually as well as together, and running the results on a gel. Are you running sufficient DNA to see a band?

Thanks so much phage434,

I appreciate your timely response. I did measure the DNA and I have about 90ng/ul. I did not run it on a gel prior to digestion. I am trying to see if I have the right recombinant plasmid by doing double digestion. I also used a metaphor gel to increase DNA resolution. I did not see any band and that is why i am having trouble. Thanks again,

[scolix]

It might be difficult to see such a small fragment. You could see it if you optimise it. Easier to sequence it.

[phage434]

I would strongly recommend running a gel with undigested and single digested DNA. I predict that you do not have plasmid DNA at all, and that your spec measurement is misleading you.

[Bic]

Hi, I am a new user. I am trying this technic for 6 month at least. Through PCR amplification, I am trying to insert a protein with EcoRI and XbaI site in pcDNA6/V5 vector. And I always failed in the tranformation, i did not obtain any colony. So i think that i commiteed a mistake before the ligation.
I have many questions. I could do double digestion with EcoRI and XbaI directly in pfu mix (PCR mix)? because I do not purify first.
Someone do the double digestion of these enzymes not sequencial? which NEB buffer i have to use?
I dont know where in the protocol is convenient to do a purification of the DNA. I read that UV degrades de DNA in a gel purification. I hope you can help me. Sorry by my bad english!

[phage434]

If you intend to clone a PCR fragment you MUST purify before cutting the DNA. Otherwise, the (still active) PCR enzymes and dNTPs will extend the 3' end of the cut DNA and destroy the cohesive site, preventing ligation.

[Bic]

If you intend to clone a PCR fragment you MUST purify before cutting the DNA. Otherwise, the (still active) PCR enzymes and dNTPs will extend the 3' end of the cut DNA and destroy the cohesive site, preventing ligation.

Thanks for your quick answer,
other Q: after the double digest i dephosphorylate the vector, i have to inactivate the RE before putting FAL?
And for the ligation, I put the ligation mix at 4ºC overnight. Is it OK? or i have to put at room temperature too?

[phage434]

I don't know what FAL is, but if it is a phosphatase, then no, you need not inactivate the RE prior to adding it. Most of the phosphatases, however, have required co-factors not present in the typical RE buffer (like zinc), so you probably will need to add the appropriate buffer.

I would recommend 10-30 minutes at room temperature for the ligation, but 4C overnight will certainly also work. Normal ligase bufffer (not quick ligase). 20 ng of vector and 1 to 3 molar equivalents of your insert in a 10 ul reaction would be where I would start. Low concentrations of vector favor recircularization rather than concatamers.

Something you did not mention was the (required) overhangs of your restriction site on your primers. I'd recommend 6-8 bases.

Do controls on your transformation efficiency. Transform with 10 pg of a common vector such as pUC19 -- you should get hundreds of colonies with cells having good transformation efficiency.