Hack a Taq

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Now that there are several DIY PCR machines available, the next challenge was to find a reasonable source for Taq polymerase. Also, as a side-project for the BIO-DESIGN for the REAL WORLD at EPFL, we wanted to see if we can make the "wet" part of DIY PCR more accessible, so that PCR could be used as a coliform bacteria detection method from water samples.
PLEASE NOTE that to use genetically modified bacteria, you will need to check and comply with your local/national regulations.
This is a happy confluence of several items on the to-do list, and the process will be documented on biodesign.cc.


EDITING IN PROGRESS!!


Objectives

1. test the Wild OpenPCR machine (gaudilabs)
2. hack the Taq isolation protocol from Open Biotechnology, and see if we can make and purify the enzyme with alternative reagents from the consumable reagents sold there (hackteria open source bioart...)
3. test out some PCR primers to detect E. coli (biodesign for the real world)


Techniques Learned

  • PCR
  • agarose gel analysis of PCR products
  • DNA extraction from E. coli
  • Taq protein extraction


You will need

We will try to make this list modular, so that you can do each part independently.

Equipment for

agarose gel electrophoresis
  • agarose gel box
  • weighing scale
  • measuring cup for volume measurements (preferably metric system)
  • pipettes
  • power source
  • camera


PCR
  • PCR conventional machine
  • Wild OpenPCR machine
  • pipettes


Taq protein production and extraction
  • heatable water bath (or a pot on a stove)
  • water boiler/pot
  • thermometer
  • vortex
  • magnetic stir plate
  • magnet
  • shaker at 37C
  • an erlenmeyer flask
  • a bottle with not-so-wide neck that can resist pressure cooking
  • bunsen burner/ spirit lamp (for bench sterile technique)
  • pressure cooker (for media sterilization)
  • electric or gas stove/cooking surface
  • weighing scale



Consumable materials

  • bacterial culture tubes
  • PCR tubes
  • pipette tips

cryotubes

Reagents

Bacterial Transformation
  • LB
  • competent host bacteria (E. coli DH5zlpha)



PCR



Protein Production



Protein Extraction



Agarose Gel Electrophoresis



Making Bacterial Stocks




Protocols

From Open Biotechnology

pOpenTaq datasheet and protocol PCR master mix protocol protein extraction buffer protocol

Colony PCR for E. coli genomic sequences

Boiling miniprep protocol

Isolation of Taq expressed in E. coli

Workshop Details

5 April - Optional pre-workshop day at EPFL
12:15pm (EPFL SV3.615) meet and work with the students of BIO-DESIGN for the REAL WORLD.
17:00 pm (EPFL) PUBLIC THESIS DEFENSE - Highthroughput 3D culture and analysis of stem cell differentiation (room and official title coming soon)
6 April
10:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market
7 April
11:00 am (m1 metro, Flon)


12 April - Optional pre-workshop day at EPFL
12:15pm (EPFL SV3.615) meet the students of BIO-DESIGN for the REAL WORLD.
13 April
10:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market