my colonies don't have the insert, how is this possible? - (Jul/07/2009 )

I get nearly 10 colonies on my agar plate after ligation and transformation, but none of them have the PCR product insert. In fact the extracted plasmid is the same size as the empty plasmid.

I cut the correct band on the gel after double digestion, but I've been told maybe one of the enzymes didn't digest well and that is why the plasmid re-ligates to itself during ligation.

I suspect my digestion, ligation and even my cells, don't know which step is my main problem.

Curtis on Jul 7 2009, 08:19 AM said:

You need controls at every step, now that it did not work the first time.

If double digest continues to give you troubles due to inadequate cut and self-ligation, you should add dephosphorylation (Shrimp Alk Phos) step.

where can i find info about this shrimp alk phos? any protocol?

my friends suggest doing single digestion step by step, would that help also?

Hey Curtis,

try to use this SAP protocol from Roche that I have attached in this reply. Do you know what the size of your insert is? If your empty plasmid and your extracted one are of the same size, then perhaps your vector re-ligated with itself or you don't have enough insert present to be ligated with your vector.
What restriction enzymes did you use? Have you check whether they have certain Buffer need for optimum level of activity?

Curtis on Jul 8 2009, 08:21 AM said:

Make sure your enzymes are compatible for double digestion... http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

What insert to vector ratio are you using?

We use antarctic phosphatase from NEB when dephosphorylating the vector.

Is it possible your gene insert could be toxic to whatever you're transforming into?

Are you digesting your insert with the same enzymes? Are the blunt end or are there overhangs?


There are a number of issues that can lead to a vector re-circularizing empty.

thanks for your replies,

my enzymes are HindIII and NheI. I am using fastdigest protocol and enzymes from Fermentas and I have tried many other temperatures and times but still no good result.

both enzymes cut the plasmid 80-90%.

the only thing that I am worried about now is that maybe my overhang nucleotides are not enough for fastdigest. I have ordered conventional enzymes now, waiting to be delivered. I have 2 nucleotides for HndIII and 5 for NheI. I've tried many different T4's and buffers because I thought maybe mine are spoiled already. the pcr size is 1,5 kb, and the plasmid is phMGFP 4.7 kb.

Curtis on Jul 16 2009, 01:11 PM said:

One thing you can do to test whether the ligation is working properly is to use the ligation product as template for a PCR using primers outside the cloning sites. If you get a correct product, that would point to a proper ligation, but something occurring during transformation. If you don't get the correct product, there's a problem with either the ligation or the digestion.

If the vector is cut with two enzymes that don't generate compatible ends, there's no need for phosphotase treatment. How did you isolate your vector after digestion? Did you gel-purify the restriction digest?

Your colleague is right -- if one of the enzymes wasn't working, or wasn't working well enough to cut your prep to completion, you'd have some singly-cut vector in your ligation, which could re-circularize during ligation. Or, if you didn't clean your vector prep after digestion, there could be circular plasmid that escaped digestion altogether.

In the either instance (enzyme(s) not cutting to completion, or vector prep contains uncut circular plasmid), I guess I would expect way more than 10 colonies, though...

Note also that, depending on the manner in which you aquired your insert, if you didn't clean your insert digestion properly, there could be little pieces of DNA (clipped from the ends of your insert if it was a PCR product, for example) left in your insert prep. These can ligate with your vector, allow circularization, and the plasmid thus created would likely be indistingushable from empty vector on the basis of size alone.