Colony lysis procedure for riboprobe synthesis

Description

This protocol starts with a single bacterial colony and makes PCR generated template for in vitro transcription reactions. The PCR reaction can also be used on plasmid DNA at 5ng/ul to generate probe template directly instead of digestion, etc.

Method

Colony picking and lysis

Colony Lysis Buffer

  • 1% Triton X–100
  • 20mM Tris pH 8.0
  • 2mM EDTA pH 8.0

Pick 6 single colonies (leave some on the plate for growth later) and place each into 25ul of Colony Lysis Buffer in a 96well PCR plate. The best technique is to smear the cells onto the side of the well. Seal plate with sealing mat or a foil sealer. (NOTE: When using clone specific primers you can pick a single colony since the primers will serve as the quality control). I use regular sterile pipet tips for picking colonies.

Heat in the PCR machine at 95C for 10min, cool on ice. The DNA is now ready for PCR.

PCR Reaction

To generate probe template, perform PCR with clone specific primers (preferred) or promoter specific primers (T3, T7, SP6), typically using a 25ul volume PCR. Below is the procedure for using promoter primers on BMAP clones (pT7T3D-PAC vector). Adjust to the established PCR conditions for clone specific primers (i.e. conditions used for primer design).

PCR Recipe - 25ul
Item Volume
Qiagen Tag Buffer 2.5ul
10mM dNTPs 0.5ul
10uM forward primer 1.25ul
10uM reverseT7 primer 1.25ul
Qiagen Taq Poly. 0.125ul
Water 18.375ul
Colony Lysate 1ul
PCR Conditions
Temp Time Cycles
94C 3min -> 1
94C 40sec |
48C 40sec | -> 35
72C 2min |
72C 5min -> 1

Run 5ul on a 1.2% agarose gel. Select one of the six that represents the expected product and amplified well. Usually all 6 or at least 4 of the six agree in size so those are the ones to pick from.

In vitro transcription

Use 10ul–12.5ul of the PCR product directly in a standard 20ul in vitro transcription reaction to produce DIG labeled probe (Lab/Roche protocol). Probe is column purified, quantified and diluted to a 20X concentration for storage at–80C (whole-mount protocol).

We have found using the PCR product directly results in excellent probe without any degradation. Using PCR cleanup columns or phenol/chloroform extraction is not necessary.

Materials

  • Reagents
    • Triton X–100
    • Tris pH 8.0
    • EDTA pH 8.0
    • PCR reagents
    • PCR primers
  • Solutions

Troubleshooting

Typically at least 5 of the colonies result in a PCR product and these will all match in size. If there is disagreement between sizes, especially when using promoter specific primers, it is recommended to discard the plate and find a new clone to try. When using clone specific primers and no product results, trying the PCR one additional time at a slightly elevated temperature (add 2C to the annealing temperature) may result be successful. Repeating more than once has proved useless.

Discussion

References

Attribution

Metadata

  • Date created: 2002–10–10
  • Date updated: 2009–02–17
  • Version: 1

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