molecular cloning gone bad

[noya12]

Hello everyone,I am in the need for desperate help after failing to clone a 2.3 kb insert to a 3.5kb target vector for a few months now.

 

This is what I have done so far:

1. Run a PCR on the gene from a library (on a plasmid source)

 

 

primers with restriction sites for XbaI and SpeI corporated.

 

2. digestion with the restriction enzymes of the very nice PCR product, and ligation to the target vector. This ligation (ratios 1:3 1:6 1:10 vector:insert) did not work no matter what I did (with and without CIP, and with a sequential cut of the vector). The target vector was verified to be linearlized

 

3. Assuming the PCR product does not cut efficiently I subcloned it into an intermediate vector Next, and cut out (step-wise) the insert. Now I was sure that the insert is cut at both ends and has the right overhanging sequences for insertion to the vector. But again, the ligation did not work (different T4 ligases, different ratios,different compatents). The target vector was also verified to be cut with both of the enzymes.

 

4. desperately I now ordered new primers with different restriction sites.

 

Most of the time I get no colonies at all and sometimes I do get some colonies both on the plate with only the target vector as well as the one with the ligation mixture but none of those is positive for my insert.

 

Can anybody offer me a piece of advice on how to proceed?

 

 

 

[Kneeanderthal]

Something similar happend to me and it was due to the toxicity of the insert (assuming your gene is toxic). If is a high copy number vector, leakage could make enough protein to kill your cells in a selective manner. I would try incubating the plate with transformants at a lower temp (try 30 C, even room temp 25 C) instead of 37 C.

 

Hope this helped some!

Edited October 23, 2012 by Kneeanderthal

[CharonY]

A simple test (provided the ligation mix is concentrated enough) is to run the ligation on a gel, to ensure that it worked.

Alternatively a few controls, such as re-ligated vector, test for competence of cells (it appears that you have done this one already) etc. could be run, just to be sure that all the components are working.

If everything works out fine, there is a good chance that Kneeanderthal is correct, the insert maybe harmful to the cell. Depending on what you want alternatives to temperature are using low-copy plasmids are a different host.

[Greippi]

How did you calculate your ligation ratios?

[noya12]

thank you all for the answers.

I do not belive my insert has a toxic nature to it. I already expressed it once in a different vector before.

I calculated the ratios both by running a sample on a gel and compering the intensities of the bands (taking the differnet sizes in mind) and both using nano drop to calculate the DNA concentration and again calculating it to be 1:3,1:6,1:10 in favor of the insert...

I ran the ligation mix and it did look alittle bit different when the insert was added.alternativly I prepared some mini-preps for a few colonies which all contained my empty target vector which ran different from the source but did not cut the insert out using the restricition enzymes...

[Greippi]

Very mystifying!

 

How long is your insert/what's the total size of the vector? What cells are you using?

[noya12]

indeed.it never happened to me before. it came to a point where I replaced all the purification kits and even asked for new DDW filters....it's a 2.3 kb insert to a 3.5kb vector.also tried with a 12kb vector. using DHs-alpha compatents.