So your PI is correct that with touchdown PCR you will often get some signal that may or may not be specific. You can sort through it if you know the precise size of the predicted product and just ignore other bands but it is an issue if you don't.

As my PI would say "you can't rush quality".

Choose a few colonies & streak them out on a new plate and also set up an overnight liquid culture (with selection). Use a plasmid prep, then set up your PCR. Might even need to linearize the plasmid using a restriction digest (make darn sure that the enzyme doesn't cut in your insert).

Good luck!

Hi Katie A S Burnette so you don't do colony PCR at all?

If I understand correctly, you wouldblindly pick several colonies, grow them, isolate plasmids and from those did PCR and/or restriction?

My PI says that speed is the most important. The problem is, when it doesn't work, which in my case unfortunately doesn't lately.

Hi there. Touch-down is perfectly compatible with colony PCR. We regularly perform multiplex colony TD-PCR (at least 5 targets). It is not related to cloning though. You could add PCR enhancers and hot-start polymerase to suppress non-specific amplification and increase the detection rate.

Our standard enhancer mix is:

300mM Tetrapropylammonium chloride, 2% DMSO, 0.05mM Hydroxy naphthol blue disodium salt (final concentrations in PCR).

Wouldn't it be simpler to use primers targeting the vector sequence either side of the cloning site? Then there would be no issue of conditions, except possibly extension time.

Hi Andrew Jenkins ,

unfortunately, we clone 6+ kb. Last time I tried to amplify 5 kb or so with OneTaq from NEB, but it didn't work (although it could be because of primers, I haven't checked again yet).

There are at least two reasons why you should use proofreading polymerase and not Taq for that purpose. First, Taq has a very high error rate, and second, the PF enzymes perform very well in Long-range PCR. Some can amplify >30kb. The last time we did cloning we amplified 6.5kb plasmid with this enzyme https://eurx.com.pl/product/e2960/

Hi Ivan Nikolaev Ivanov ,

thank you for your insight. However, now I'm talking about colony PCR. For cloning, I use of course proofreading polymerases.

If your insert is as big as you say, why not just do a rapid plasmid prep and run a gel?

Tomáš Hluska I never do colony PCR, and certainly not from the original selection plate. The rate of false positives (and false negatives) mean it's usually just a waste of time.

Yes, I would randomly pick a few colonies to streak out & set up a liquid prep. I'd rather spend 1 day setting up cultures & the next doing a plasmid prep & PCR knowing that I can have confidence in my results.

I've never had issues with false positives (or false negatives). The data speak for themself.

If your colony PCR fails or you end up screening out false positives, I'm betting it takes you more than just 1 day to get things working.

But, I know other folks get colony PCR to work. They do set up liquid cultures or pick from a streak plate.