How to do colony PCR with Agrobacterium tumefaciens (or other difficult to lyse cells)

If the cells don't lyse well, they result in a smear instead of a band.

Example 1% agarose gel of Agrobacterium tumefaciens colony PCR:

Lanes:

1. Colony PCR with pre-boiled Agrobacterium produces the expected band size.
2. DNA ladder, NEB 2-log
3. Colony PCR with unboiled Agrobacterium added directly from the LB agar plate fails, results in streaks. This happens with the standard colony PCR protocol for E. coli with a 10 minute step at 95C. It's not enough to lyse Agrobacterium, so the PCR fails.

My standard colony PCR protocol for E. coli:

(1) Prepare 25 uL PCR reaction tubes containing ThermoPol buffer (for Taq DNA polymerase), water, dNTPs, primers, and Taq DNA polymerase. No template added yet.

[*Note, you can use other buffer and polymerase combinations. This doesn’t really matter. Like everyone else, I use Taq because it’s cheap.]

(2) Pick up a single E. coli colony from an LB agar selection plate with a sterile pipette tip, very gently re-streak onto a fresh LB agar selection plate (to keep a copy of the clone), and then swirl the pipette tip into the PCR reaction mix to use the remaining cells for DNA template.

(3) Run the PCR:

• 94C for 10 minutes to lyse cells and release genomic DNA 
• {94C for 30 sec, 58C for 30 sec, 72C for 1 min/kb} x 30 cycles to amplify product 
• 72C for 5-10 minutes, final extension 
• 4C hold 

(4) Run approximately 4 uL of PCR product on an agarose gel.

This works well for me when I’m using E. coli. However, trying this with Agrobacterium tumefaciens gives me streaks on the agarose gel and no visible product. I suspect either the Agrobacterium cells don’t lyse with 10 minutes of high heat or else they contain PCR inhibitors.

Here is my modified protocol for colony PCR with Agrobacterium:

(1) Pick a single Agro colony from an LB agar selection plate, re-streak most of it onto a fresh LB agar selection plate (so I keep the clone) and swirl the rest of the cells into a microcentrifuge tube with 50 uL of sterile ddH2O.

(2) Heat the cells at 95C (boiling also okay) for 30 minutes, then place on ice to cool.

(3) Meanwhile, prepare 25 uL PCR reactions as usual.

(4) Add 1 uL of the cells+water mix into the PCR reaction as DNA template.

(5) Run the colony PCR program. I still leave the 10 minute lysis step in my program, but it should no longer be necessary.

(6) Run samples on an agarose gel.

(7) Marvel at the clean product bands.