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ERIC PCR Protocol

Background

This SOP describes how to carry out ERIC PCR to differentiate strains based on a random PCR creating strain-variable banding patterns. This protocl is based on Ventura and Zink (FEMS Microbiology Letters 2002). Note: This protocl in theory was designed to amplify a feature of The E. coli genome, but really is just randomly amplifying the genomic DNA.

Materials

  • Instagene Matrix (Biorad #732-6030)
  • Wide bore 200 µL pipette tips (or clean scissors to cut tip off end of normal tip)
  • 1.5 mL nuclease-free microcentrifuge tubes
  • nuclease-free water or PBS
  • 200 µL PCR tubes
  • Waterbath/heatblock/PCR thermocycler capable of 56˚C and 100˚C
  • Amplitaq Gold 360 Master Mix (Life Tech/Thermofisher 4398881)
  • ERIC1R primer (5-ATGTAAGCTCCTGGGGATTCAC-3) at 100 µM
  • ERIC2 primer (5-AAGTAAGTGACTGGGGTGAGCG-3) at 100 µM
  • Nuclease/DNA free H2O
  • Waterbath and/or heatblocks set to 56˚C and 100 ˚C

3. DNA Extraction

  • Pick a single colony from a freshly prepared culture plate and resuspend in 1mL of PBS or sterile H2O in a microcentrifuge tube OR Transfer 200ul from a broth culture to a microcentrigue tube.
  • Centrifuge at ~10,000 xg for 1 minute.
  • Remove the supernatant and resuspend in 100 µL of Instagene matrix (matrix should be well mixed and use wide bore tips)
  • Mix thoroughly by vortexing
  • Incubate at 56˚C for 30 minutes
  • Mix thoroughly by vortexing
  • Incubate at 100˚C for 8 minutes
  • Mix thoroughly by vortexing
  • Centrifuge for 3 minutes at max (~16,000 xg)
  • Transfer 60 µL of the supernatant containing gDNA to a new microcentrifuge tube. This is optional and supernatant can be used directly for PCR.
  • Store gDNA at -20˚C for long term storage or 4˚C for short term.

4. PCR

  • Prepare a master mix for PCR as outlined in Table 4.1.
  • Add 1 µL of gDNA for a total reaction volume of 20 µL. Be sure to include a reaction with 1 µL of H2O as no template control (NTC).
  • Pop spin to remove any air bubbles.
  • Run the PCR using the PCR cycle parameters indicated in Table 4.2
Table 4.1 PCR Master Mix
Component Stock Concentration Final Concentration per 20µL rxn
Amplitaq Gold 2x 1x 10 µL
Water - - 5 µL
ERIC1R 10 µM 1000 nM 2 µL
ERIC2 10 µM 1000 nM 2 µL
gDNA variable variable 1 µL
Total 20 µL
Table 4.2 PCR Cycles
Cycle Temperature (˚C) Time
Initial Denaturation 95 10 min
35 cycles:
Denature 95˚C 30 sec
Anneal 48˚C 60 sec
Extend 72˚C 4 min
Holding 4˚C Hold 0 sec

4. Interpretation

Compare banding patterns across gel. If two strains are related they will share many bands, and if two strains are identical, they will have identical banding patterns.