Dear Akhileshwar Singh

How did you perforrm the colony screening? using which primers?

How big is your insert?

In my experience some times happen that some of the clones in the TOPO cloning are inserted in the opposite directions and this may be negative to PCR screening using a primer that anneal in the GOi and one other that anneal in the plasmid.

best

Manuele

Hi Akhileshwar,

I don't know all the materials you have used however I have some ideas/recommendations

1. Are you sure that your colony PCR is working - colony PCR could sometimes be tricky? I would advice to do a mini prep and to perform restriction analysis in parallel, to see if you have an insert. If you really have no insert check the following:

2. Does your forward primer have the required CACC at the 5' prime end? (if you have done this correctly your insert should be in the right direction)

3. Did you use a proofreading polymerase for the initial PCR reaction?

4. Avoid gel purification - use the fresh PCR product (as recommended in the manual)

5. Avoid using to much PCR product as this will interfere with cloning efficiency.

Good luck

this can be due to unproper ligation, as long the process is involving the ligase enzyme don`t trust this enzyme, sometimes it mess up.

Manuele Martinelli I usually pick the colony and mix it in 100ul distilled water and use 1ul of this as template in 10ul of PCR reaction.

I use the gene specific primers that was used to amplify the gene for the colony PCR as well.

My insert on an average is 1.5kb.

Dörthe Andrea Kesper I think my colony PCR technique has no issue becasue I am doing this colony PCR in parallel with other genes, where the result is positive.

But ever since I have used those genes that were amplified from plasmid, I am geting negative result.

Note- For those plasmids that I am using as a template to amplify my genes are also cloned in TOPO vector which I had successfully cloned and got positive colonies. But those genes where amplifed from the cDNA of the source organism.

Yes, I have added CACC in the forward primer.

I am using pfu polymerase for amplification

Hi Akhileshwar,

Your Primer designer and your polymerase sounds good.

However, I would still try to do the restriction analysis, because if your colony PCR work for one gene it is not guaranteed that it will work for another ( I hope I understood it correctly that you use gene specific primers).

You also said that you did a gel purification and assume you did do this under UV-light? This could rapidly induce mutations in the DNA (depending on the wave lenght) and severly reduce the cloning efficiency. And if you are really unlucky it could also mutations in the sequence were your gene specific primers are binding and thereby interfering with proper priming - for this reason it might be a good idea to do your colony PCR with the vector primers you got with the kit, to see if you can amplify something with these primers (if you haven't done this already).

If you have to do gel purification because you have more than one band avoid prolonged exposure to UV light - however if you have a single band use the fresh PCR product directly.

And as the Insert is relativly big it might also help to prolong the incubation time.

Good luck

Colony PCR is tricky to get right. Sometimes the secondary structure of the plasmid makes it difficult to amplify any PCR products, regardless of size. I'd recommend finding an inexpensive single-cutting enzyme and do a restriction digest to linearize first. (Make certain that the enzyme does NOT cut anywhere in your insert). Grow up an overnight liquid culture with selection, purify the plasmid, set up a restriction digest (ideally with a heat-inactivated enzyme so you don't need to do another purification), then use that for PCR.

As my advisor would say "You can't rush quality". Sometimes you have to do the longer protocol to get your data.

Good luck!