Cloning long fragment...got lots of colonies with vectors that have no inserts.. - (Oct/07/2010 )

Hi, All,

I'm first time here and I'm really frustrated by cloning now.
I've been trying to clone a 4kb insert into a vector for almost 2 months.

What I did is....

1) I cut both plasmid and insert with KpnI-HF and XmaI.
2) Purify the digested product with Quiagen purification kit.
3) Ligate them using Invitrogen T4 ligase using insert:vector ratios like 3:1 or 5:1.
4) Transform the ligated product to DH5-alpha competent cells.

I always do a negative control for transformation and each time I get no colonies on negative control plates but with lots of colonies on the plate transformed with the supposedly ligated vector.

The weird thing is if I take 16 colonies. All of them only have vectors without insert.

The question is I don't know how this is possible because the cut vectors should not reclose to themselves and produce vectors without inserts.

I've tests to make sure both enzymes work: I tried to cut the vector with KpnI-HF only, with XmaI only, with both and with none of them All of them produce a clear band at the right size. the last one has a supercoil and the others are linearized forms.

In addition, I've made sure that the enzymes should be able to digest all of the vectors I added in so there should not be undigested vectors in the product I used for transformation.

Anyone got any clue about what's happening here? Please give me some suggestion!! Thanks a million!!!

Allison

Lots of missing information. Where does the insert come from? What's your vector? How are you cutting? Ligating? Transforming? We need to know lots more to give advice on potential problems.

phage434 on Fri Oct 8 02:55:48 2010 said:

The plasmid is a the pCFJ178 vector derived from Jorgensen lab: http://sites.google.com/site/jorgensenmossci/vectors

The insert is the gene end-1 from C. elegans.

The cutting is done by double digestion with KpnI-HF and XmaI, 37C 1hr, 1X BSA and NEB Buffer 4.

Purify the DNA by using Qiagen purification kit.

I've tried insert:vector ratio 5:1 or 3:1 for ligation using invitrogen T4 ligase. and tried to ligate at 15C overnight, or 15C over weekend, or RT 1hr.

The transformation is done by adding 10ul of the ligated product, 30mins on ice, 20sec heat shock at 42C, incubate at 37C for 1hr and then plate them on LB/amp agar plates.

That's basically all I have done. Please let me know if you need more information. Thanks in advance.

Allison

let start
0- how long is your digestion? Was enough time given to complete the digest?

1 - did you gel purify the DNA fragments?

1.1 - was the vector dephosphorylated?

2- Who long did it take for you to excise the DNA band under UV light? UV exposure should be measured in seconds. Add 1mM guanosine (final concentration) to the gel to act as a UV blocker.

3- how far apart are the XmaI and KpnI site on the vector? If the sites are too close (less than 6bp apart) the enzymes won't cut.

4- The ratios use.. are they ratios in mols (number of molecules)?. The ligation ratios are in mols not mass.

5- Both T4 DNA ligase and ligase buffer go off very very easily. Does your ligase buffer have a strong smell? (You are smelling DTT). If ligase buffer does not have a horrible pungent smell, it is bad and has been oxidized. Get new buffer. Aliquote new buffer into several smaller tubes.

6-Run a some of ligation mix onto a gel (use the narrowest smallest well you can find). You should see high molecular weight bands, which are your ligated products. If you do not see these bands.. the T4 ligase is probably bad. Are any of your labmate experiencing problems with ligation? Buy only small vials of T4 ligase, the enzyme amazingly does denature at -20C given enough time.

0- how long is your digestion? Was enough time given to complete the digest?

-->37C 1hr should be enough according to the manual. Sometimes I let it stand for a little more than 1hr.

1 - did you gel purify the DNA fragments?

-->I use Qiagen purification kit to purify digested products.

1.1 - was the vector dephosphorylated?

-->How do you check this?

2- Who long did it take for you to excise the DNA band under UV light? UV exposure should be measured in seconds. Add 1mM guanosine (final concentration) to the gel to act as a UV blocker.

-->I'm using cybersafe so it's actually blue light, which I don't think would damage DNA.

3- how far apart are the XmaI and KpnI site on the vector? If the sites are too close (less than 6bp apart) the enzymes won't cut.

-->There is about 50bp apart. Should it be ok?

4- The ratios use.. are they ratios in mols (number of molecules)?. The ligation ratios are in mols not mass.

--> it's mole ratio.

5- Both T4 DNA ligase and ligase buffer go off very very easily. Does your ligase buffer have a strong smell? (You are smelling DTT). If ligase buffer does not have a horrible pungent smell, it is bad and has been oxidized. Get new buffer. Aliquote new buffer into several smaller tubes.

6-Run a some of ligation mix onto a gel (use the narrowest smallest well you can find). You should see high molecular weight bands, which are your ligated products. If you do not see these bands.. the T4 ligase is probably bad. Are any of your labmate experiencing problems with ligation? Buy only small vials of T4 ligase, the enzyme amazingly does denature at -20C given enough time.

for 5. and 6., I'll check with this but I actually used new T4 ligase. I just bought the new T4 ligase 2 month ago right before I started my cloning. Right now there is only me doing cloning in my lab.
Will it degrade so fast in only 2 months?

Thanks!! I think ligase might be a problem. I'll double check.
But one thing I still don't understand is how I can still get colonies with vectors w/o inserts.... Does anyone have any clue?

thanks!!

allison

So, is the insert a PCR product? Are restriction sites added to the ends of the primer? Are extra bases added to allow cutting?

phage434 on Fri Oct 8 21:37:20 2010 said:

Yes the insert is a PCR product and both restriction sites are added and there are extra bases added to allow cutting.

Do you purify the PCR product prior to cutting?

It really would be much easier if you explained in detail EXACTLY what you are doing, instead of giving general hints.

The biggest problem I see is that you're "purifying" the digested DNA with a Qiagen kit. This kit will recover all DNA, and not discriminate between digested and undigested DNA. Thus, you're retaining uncut vector in your sample, and it's transforming the recipient, resulting in transformants containing empty vector. You need to gel purify your digested DNA to separate cut from uncut.

If you're getting lots of empty vector colonies it implies to me that you are not getting complete digestion, I usualyl try and digest for 2-3 hours rather than 1 hour, but I am using Promega enzymes which reccomend 2-4 hours. try a longer digest?

Phosphorylation shouldn't be neccecery as you are using 2 different enzymes, so I don't think you need to worry about that.

I saw someone else on here reccomend using an additional restriction enzyme, after the ligation, that cuts at a site in-between the Kpn and Xma sites in the vector, but not in the insert. That way any empty vector present will be cut, but anything with an insert in won't be.