Colony PCR Question - Get the band i want but mini-prep shows no plasmids! (Nov/16/2009 )

So i tried colony pcr for the first time and used a simple protocol online complete with a lysis buffer recipe.

Used 4ul of supernatant to do PCR and I got a majority of the colonies picked to generate a band the approximate size of my insert.

When i culture the bacteria and then perform a mini-prep and double digestion to release the insert, there appears to be no plasmid at all when the gel is run. Even more, i check the concentration and all of the potential positive clones have a concentration.

How can I get a possible amplification of my insert if I don't have plasmid present? (How did the DH5 grow in the first place???)

Is the concentration just too low but the insert is there?
Is there some kind of contamination?
Did my unligated insert manage to transfect and when the DH5 lysed, was still able to serve as a template for PCR?

Any help is appreciated!

Can you be more precise about your colony PCR protocol?

Based on what you had written, probably the plasmid concentration is too low to be visualized...

adrian kohsf on Nov 16 2009, 08:28 PM said:

So i picked 4 colonies with a sterilized toothpick normally used for yeast
Then i swirled the toothpick in 30ul autoclaved water in 1.5ml eppendorf tube and saved 10ul to innoculate LB+Amp overnight
The remaining 20ul was treated with 50ul lysis buffer for 10minutes at 95C then spun at high speed for another 10 minutes to pellet the cellular debris.
4ul of supernatant was used for PCR

http://www.cbs.umn.edu/labs/perry/Protocols/bactPCR.html

Lysis Buffer
# 1% Triton X-100
# 20mM Tris*Cl pH 8.0
# 2mM EDTA pH 8.0

This is my protocol for colony PCR and it has worked very well.

Using a 200 ul pipette tip, pick up a small portion of a colony and swirl it in 50 ul of sterile water. Use 1 ul of that as template in PCR. No lysis buffer or anything. Have obtained very good bands, depending on the reaction, with little nonspecific binding, and products up to about 5 kb. I use Phusion polymerase from NEB the majority of the time.

Hasn't failed me yet. Of course, it could be more difficult with different bacteria. I work with a Gram negative enteric.

I have also done it with E. coli for screening of clones. It has worked with both high (pBluescript) and low to medium (pBBR1, pGP704) copy plamsids.

Also need to add that with Phusion, rather than doing the initial 98 C for 30 seconds, I extend it to 5 minutes, then cycling is normal after that.

I do exactly what fishdoc does, except with regular Taq (NEB's 2X master mix) because I'm just sending the amplicon for sequencing (not cloning the insert), and regular Taq works fine and is cheaper. Over the last year, I've probably screened 1000 colonies of about 20 different species from clinical isolates, and haven't had a failed PCR yet.

I quote Adrian; could be that the amount of plasmid you run on the gel was just too low. if baki can grow on selective medium, the plasmid should be there (of course unless your primers amplify genomic coli DNA and you ampicillin is degraded )
If @ spectrophotometer you see DNA (and if it's not RNA carryover, depending on the miniprep protocol you use), just load a hundred ng (or more) of uncut plasmid on the gel and you should see it. if your concentration is low, it could be that your miniprep did not work (or you lost the pellet).

rocketfan86 on Nov 17 2009, 01:28 PM said:

As far as I understand for a colony PCR, lysis buffer was never used, but the bacteria is suspended in water and/or directly put in master mix and do PCR. Your method is more or less like a extraction protocol. Moreover, 20ul is too little if you want the plasmid to be seen obvious by using your method in agar. If you still want to "see" the plasmid, use a plasmid prep method. There are few commercial kits available. I use Promega PureYield™ Plasmid Miniprep and it works excellent for me. They used to give free trial kits but I not sure whether the offer is still valid.

All the best.