Hack a Taq

From Hackteria Wiki
Revision as of 11:55, 7 April 2013 by Dusjagr (talk | contribs)
Jump to: navigation, search
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-hack-a-taq documentation. Once the session 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
  • IDEXX coliform / e.coli detection

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)


DesktopHDDCentrifuge.jpg


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 - we discuss the background on why we first chose some primers for the tuf gene on biodesign.cc.


Protein Production
  • LB media
  • 2xYT media (this link explains the different types of media, and their applications)
  • 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



Isolation of Taq expressed in E. coli

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

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.

Typical PCR conditions with Taq

Here is a place to start from the NEB site.

Agarose Gel Electrophoresis

Nice protocol on Addgene.


IDEXX Coliform detection

We tested the Colilert®-18 from IDEXX. The reagent we received loooong ago from some peeps at EAWAG. Diluted some samples of Rosetta with PBS, various dilutions 1, 1:20, 1:400, control PBS. Incubated in a cardboard box above the hot-water piping in the hallway overnight. Nothing turned yellow.... yet....

Details

What to bring, Where to stay

For the Hack-a-Taq weekends, we will be preparing materials outlined above.
The idea is for you to read what is on this page, get inspired, and do bring your own ideas and materials for the session!
Come for parts of the day, come to all the days.

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 and Meeting Points

5 April - Optional pre-workshop day at EPFL
14:00pm (EPFL SV3.615) meet and greet.
18:00 pm (EPFL BM5202) PUBLIC THESIS DEFENSE - Highthroughput 3D culture and analysis of stem cell differentiation (room and official title coming soon)
6 April
11:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market -
We are running late this morning - for any questions, please contact Sachiko (at) biodesign.cc
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


New Hackteria GelBox

During the pre-Hack-a-Taq night on thursday, we went to FabLab Luzern to make some gadgets to be used in the lab.


HiroKouri Gel Carrier Souvernir

Hackteria GelBox v0.1

GelBox assembly.jpg

GelBox glueing.jpg


Testing the GelBox

We gave it a try using GeneRuler 100bp. To get more Voltage we connected 2 powersupplies in series. Let it run for ca 50 min and then stained with MethBlu overnight. Seems we overstained it quite lot, but washing out the color from the gel makes the ladder visible again. Also seems we ran it to far (long)

GelBox setup.jpg