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.

Publication: GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

Colleen A. Kellenberger, Stephen C. Wilson, Scott F. Hickey, Tania L. Gonzalez, Yichi Su, Zachary F. Hallberg, Thomas F. Brewer, Anthony T. Iavarone, Hans K. Carlson, Yu-Fang Hsieh, and Ming C. Hammond

PNAS April 28, 2015 112 (17) 5383-5388; published ahead of print April 6, 2015 https://doi.org/10.1073/pnas.1419328112

Links

• PNAS article
• PubMed ID: 25848022
• PubMed Central ID: PMC4418906

Highlight

• We discovered a riboswitch subclass that prefers to bind cyclic AMP-GMP (cAG). Previously, it was assumed to bind cyclic di-GMP (cdiG) due to sequence similarity.
• The anaerobic bacterium Geobacter sulfurreducens contains an operon regulated by the cAG-binding riboswitch.
• Geobacter sulfurreducensand also produces cAG.
• We developed a method to detect cAG inside living cells, a useful biotechnological tool to study cAG signaling.