Hi there,

One possibility is that you have ligated products on your plate, but not in the clones you picked. When spreading bacteria one also spreads diluted ligation onto the plate so when the clones are picked they could perhaps appear positive by PCR but are in fact "contaminated" by what's on the plate. You can control for this by scraping part of the plate without colonies and see if you get a product. Maybe carefully picking in the middle of colonies will help reduce this.

Also, XhoI SalI sites have compatible ends so you want to de-phosphorylate the vector, if you're not already doing so, to reduce re-ligation background colonies. Also use a self-ligation (vector only) control to check for background. Run your PCR on a few self-ligation colonies to control for non-specific amplification on your reagents or genomic DNA.

Hi Fernanda,

do you run a negative control (i. e., all PCR components but no colony)? It is possible that you have a problem with contamination of one of your PCR components with pPICZalfaA vector. Another explanation might be that the primers are able to amplify some genomic sequence. This might happen if you chose your annealing temperature (TA) too low (so that the PCR conditions are not stringent enough). What happens if you would increase the TA?

Best regards

Hi Fernanda. One possible scenario that I have seen happen before is that if you are using toothpicks, you may be adding contaminating DNA into your PCR reaction that acts as the template for your PCR. This is especially likely if you are trying to clone a genomic DNA fragment and the toothpicks have been used to pick cells/colonies before. If this is the case, try using a disposable pipet tip instead. I hope it helps.

Hi there,

One possibility is that you have ligated products on your plate, but not in the clones you picked. When spreading bacteria one also spreads diluted ligation onto the plate so when the clones are picked they could perhaps appear positive by PCR but are in fact "contaminated" by what's on the plate. You can control for this by scraping part of the plate without colonies and see if you get a product. Maybe carefully picking in the middle of colonies will help reduce this.

Also, XhoI SalI sites have compatible ends so you want to de-phosphorylate the vector, if you're not already doing so, to reduce re-ligation background colonies. Also use a self-ligation (vector only) control to check for background. Run your PCR on a few self-ligation colonies to control for non-specific amplification on your reagents or genomic DNA.

You suppose to run negative control for PCR every time(using water as template).So you will be sure that PCR components are not contaminated. And should run PCR by using vector specific primers.So you can distinguish between self ligated vectors and true transformants. Contaminates or ligation mixture on plate could be a cause for false positives.

As others already suggested having negative control is important. you need to check the PCR components also. there may be contamination somewhere there. vector specific primer can also help you. always use positive and negative controls and check possible issues one by one. all the best.

What can happen (and frequently does) is a cross-contamination from the DNA you are working with. PCR is very sensitive, so if you pipette or any buffer contains only traces, you will find it.

What you can do to avoid re-ligation of you vector is to dephosphorylate it after the restriction digest.

Hello guys, thank you very much! I always make a negative control for my PCR reactions, but I'm afraid of PCR be amplifying insert leftovers in the ligation reactions. I don't know if it's possible. However, I think I will try a vector dephosphorilation. Does anyone has a dephosphorilation protocol to give me, please?

I was having good vector dephosphorylation results with this protocol from Thermo Scientific (please see attachment).

Hello everybody, I need to update the topic! I tested the primers in a vector PCR, tried several "combinations". The combinations were: specific forward primer (insert) + specific reverse primer (insert); forward and reverse primers of the vector; specific reverse primer (insert) + forward primer of the vector. I made a negative control for each reaction also. The only combination that didn't give an amplification was the last one... it's seems that the other primers are amplifying free inserts and give me a false-positive colony PCR! Now, I think I will try to do a new colony PCR with new primers and then a ligation with a desphosphorylated vector. What do you think?

Too bad that I've read this thread recently. Indeed false positives are always happen, if you are using primers, both specific for only vector or only insert. The reason is as you are already correctly assuming that with these combination of primers it will always amplify non-ligated linearized insert or vector DNA. When designing primer for colony PCR, the sequence of interest has to be flanked by a vector specific primer and an insert specific primer. Dephosphorylation is a good idea as you are using XhoI and SalI which generate compatible sticky ends.

Good luck :)!

Thank you everyone for your kind contribution. Recently I faced similar situation, and would like to share it, so that someone else would be benefited.

Vector : pet16b

Insert : 1308 bp

RE : NdeI and XhoI

Following the double digestion and ligation, I got many colonies and my colony PCR (with Gene-specific primers) indicated 4 colonies as positive out of 10. I sequenced all of them and failed.

After having read the discussions, I repeated the PCR with purified plasmid of these 4 colonies individually with

1)     Pair of gene-specific primers

2)     Pair of vector-specific primers

I got the false positive (! As already sequencing is failed) data for PCR with gene-specific primers, but vector-specific primers did not show any bands, implying reliability of using vector –specific primers (Fig. 1).

Next, I picked further colonies from the same plate, and along with the plasmid I already ad purified, I performed the PCR with one gene specific forward primer and one vector-specific-reverse primer. Data showed no amplification (Fig. 2).

Based on this, I suggest that combination of gene and vector specific primers is best to screen colonies, to avoid unnecessary confusion.

Hey, guys, I got the same for pDONR207. I Used both primers of the insert and also tried the other combination (Forward primer of the pDONR207+Reverse primer of the insert). in both the cases the results were positive but when I send it to the company, they couldn't purify the plasmid even? 

Any possible reason? Thanks in anticipation