Hack a Taq

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Revision as of 21:35, 31 March 2013 by Gaudi (talk | contribs) (PCR)
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Hack-a-taq.jpg


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.
, which can be found here for Switzerland: BAG and here

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!!
This is the pre-workshop documentation. Once the workshop is over, missing details will be added.


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

  • 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 (Urs)
  • weighing scale
  • measuring cup for volume measurements (preferably metric system)
  • pipettes
  • power source
  • camera
Agar 4.jpg


PCR
  • PCR conventional machine
  • "Wild OpenPCR" machine (Urs)
  • "My Personal OpenPCR" machine
  • pipettes
ConvetionalPCR.jpg
WildOpenPCR.jpg
MyPersonalOpenPCR.jpg
Conventional PCR Wild OpenPCR My Personal OpenPCR



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
  • desk top centrifuge (Urs)


Consumable materials

  • bacterial culture tubes
  • PCR tubes
  • pipette tips
  • cryotubes


Reagents

Bacterial Transformation


PCR
  • master mix
  • PCR grade water
  • dNTP
  • MgCl2
  • commercial Taq
  • buffer concentrate
  • forward and reverse primers


Protein Production
  • LB
  • 1mM IPTG
  • chloramphenicol (34ug/ml final)
  • ampicillin (100ug/ml final)


Protein Extraction
  • Open Biotech Lysis Buffer
  • lysozyme
  • CaCl2
  • DNAse
  • Ammonium Sulfate


Agarose Gel Electrophoresis
  • Nile Blue (or methylene blue) for visualizing DNA bands
  • 100bp DNA Ladder (size reference)
  • loading buffer (to make the sample settle in the well)
  • running buffer


Making Bacterial Stocks

50% glycerol sterile

Protocols

From Open Biotechnology


Colony PCR for E. coli genomic sequences

Boiling miniprep protocol = add water and boil

from the bio.net thread from 1997

Or, just drop the bacteria/colony into the PCR tube and start the PCR

Also take a look at this well-documented resource Isolation and Purification of Total Genomic DNA from Gram-Negative Bacteria.


Isolation of Taq expressed in E. coli

We will be comparing the pOpenTaq protocol provided by Open Biotechnology, with another protocol provided by the protein expression core facility at EPFL. Taq_polymerase_isolation_from_E_coli.pdf


Workshop Details

What to bring, Where to stay

For the workshop itself, we will be prepare the materials as outlined above.
If you are coming from out of town, it would be great to know which nights you will need accommodations. It would be best if you can bring a camping mattress and a sleeping bag in addition to change of clothes and toiletries. Please contact Sachiko to set up arrangements: sachiko a t biodesign.cc

Dates and Times

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