Difference between revisions of "Hack a Taq"

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Line 1: Line 1:
 
[[File:Hack-a-taq.jpg|none|400px|]]<br><br>
 
[[File:Hack-a-taq.jpg|none|400px|]]<br><br>
 
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 [http://www.biodesign.cc 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. <br>
 
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 [http://www.biodesign.cc 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. <br>
'''PLEASE NOTE''' that to use genetically modified bacteria, you will need to check and comply with your local/national regulations.<br>, which can be found here for Switzerland: [http://www.bag.admin.ch/themen/medizin/00708/01983/index.html?lang=de BAG] and [http://www.bafu.admin.ch/biotechnologie/02618/index.html?lang=de here]
+
'''PLEASE NOTE''' that to use genetically modified bacteria, you will need to check and comply with your local/national regulations,<br> which can be found here for Switzerland: [http://www.bag.admin.ch/themen/medizin/00708/01983/index.html?lang=de BAG] and [http://www.bafu.admin.ch/biotechnologie/02618/index.html?lang=de here]
  
 
This is a happy confluence of several items on the to-do list, and the process will be documented on [http://biodesign.cc biodesign.cc].<br><br><br>
 
This is a happy confluence of several items on the to-do list, and the process will be documented on [http://biodesign.cc biodesign.cc].<br><br><br>
  
 
'''EDITING IN PROGRESS!!'''<br>
 
'''EDITING IN PROGRESS!!'''<br>
This is the pre-workshop documentation. Once the workshop is over, missing details will be added.
+
This is the pre-hack-a-taq documentation. Once the session is over, missing details will be added.
  
  
Line 19: Line 19:
 
* DNA extraction from ''E. coli''
 
* DNA extraction from ''E. coli''
 
* Taq protein extraction
 
* Taq protein extraction
 +
 +
* [http://www.idexx.com/view/xhtml/en_us/water/products/colilert-18.jsf IDEXX] coliform / e.coli detection
  
 
== You will need ==
 
== You will need ==
Line 31: Line 33:
 
* power source
 
* power source
 
* camera<br>
 
* camera<br>
[[File:Agar 4.jpg|none|400px|]]
+
[[File:Agar 4.jpg|none|350px|]]
 
<br>
 
<br>
  
 
===== PCR =====
 
===== PCR =====
 
* PCR conventional machine
 
* PCR conventional machine
* Wild OpenPCR machine (Urs)
+
* "Wild OpenPCR" machine (Urs)
 +
* "My Personal OpenPCR" machine
 
* pipettes
 
* pipettes
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
| [[File:WildOpenPCR.jpg|none|x250px|]]  ||  [[File:MyPersonalOpenPCR.jpg|none|x250px|]]  
+
| [[File:ConvetionalPCR.jpg|none|x250px|]]  || [[File:WildOpenPCR.jpg|none|x250px|]]  ||  [[File:MyPersonalOpenPCR.jpg|none|x250px|]]  
 
|-
 
|-
! Header text !! Header text !! Header text
+
! Conventional PCR !! Wild OpenPCR !! My Personal OpenPCR
 
|}
 
|}
  
Line 65: Line 68:
 
* weighing scale
 
* weighing scale
 
* desk top centrifuge (Urs)
 
* desk top centrifuge (Urs)
 +
<br>
 +
[[File:DesktopHDDCentrifuge.jpg|none|x350px|]]
 
<br>
 
<br>
  
Line 76: Line 81:
 
==== Reagents ====
 
==== Reagents ====
 
===== Bacterial Transformation =====
 
===== Bacterial Transformation =====
* LB
+
* LB media
 
* competent host bacteria (E. coli [http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html Rosetta strain] for protein production after [http://en.wikipedia.org/wiki/Lac_operon#Mechanism_of_induction IPTG induction] )
 
* competent host bacteria (E. coli [http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html Rosetta strain] for protein production after [http://en.wikipedia.org/wiki/Lac_operon#Mechanism_of_induction IPTG induction] )
 
<br>
 
<br>
Line 87: Line 92:
 
* commercial Taq
 
* commercial Taq
 
* buffer concentrate
 
* buffer concentrate
* forward and reverse primers
+
* forward and reverse primers - we discuss the background on why we first chose some primers for the [http://www.wikigenes.org/e/gene/e/960150.html ''tuf'' gene] on [http://biodesign.cc/2013/03/28/coliform-bacteria-detection-by-pcr/ biodesign.cc].<br>
 +
* Lyophilized [http://www.jenabioscience.com/cms/en/1/catalog/1425_taq_hot_start_master_lyophilisates.html Taq mix] (no cold chain needed)
 
<br>
 
<br>
  
 
===== Protein Production =====
 
===== Protein Production =====
* LB
+
* LB media
 +
* 2xYT media ([http://www.exptec.com/Reagents/Common%20Media.htm this link] explains the different types of media, and their applications)
 
* 1mM IPTG
 
* 1mM IPTG
 
* chloramphenicol (34ug/ml final)
 
* chloramphenicol (34ug/ml final)
Line 106: Line 113:
  
 
===== Agarose Gel Electrophoresis =====
 
===== Agarose Gel Electrophoresis =====
* Nile Blue (or methylene blue) for visualizing DNA bands
+
* Nile Blue or [http://en.wikipedia.org/wiki/Methylene_blue methylene blue] for visualizing DNA bands
 
* 100bp DNA Ladder (size reference)
 
* 100bp DNA Ladder (size reference)
 
* loading buffer (to make the sample settle in the well)
 
* loading buffer (to make the sample settle in the well)
Line 122: Line 129:
 
* protein extraction buffer [http://www.openbiotech.com/v/vspfiles/assets/images/e%20coli%20protein%20extraction%20kit%20for%202%20liters.pdf protocol]
 
* protein extraction buffer [http://www.openbiotech.com/v/vspfiles/assets/images/e%20coli%20protein%20extraction%20kit%20for%202%20liters.pdf protocol]
 
<br>
 
<br>
 +
 +
===== Isolation of Taq expressed in ''E. coli'' =====
 +
We will be comparing the pOpenTaq [http://www.openbiotech.com/v/vspfiles/assets/images/popentaq%20protocol.pdf protocol] and the [http://www.openbiotech.com/v/vspfiles/assets/images/e%20coli%20protein%20extraction%20kit%20for%2020%20liters.pdf protein extraction kit protocol] provided by Open Biotechnology, with another protocol provided by the protein expression core facility at EPFL:
 +
[http://hackteria.org/wiki/index.php/File:Taq_polymerase_isolation_from_E_coli.pdf Taq_polymerase_isolation_from_E_coli.pdf]
 +
<br><br>
  
 
===== Colony PCR for E. coli genomic sequences =====
 
===== Colony PCR for E. coli genomic sequences =====
Line 129: Line 141:
  
 
Also take a look at this well-documented resource [http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf Isolation and Purification of Total Genomic DNA from Gram-Negative Bacteria].
 
Also take a look at this well-documented resource [http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf Isolation and Purification of Total Genomic DNA from Gram-Negative Bacteria].
 +
<br><br>
 +
 +
===== Typical PCR conditions with Taq =====
 +
Here is a [https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction place to start] from the NEB site.
 +
<br><br>
 +
 +
===== Agarose Gel Electrophoresis =====
 +
Nice [https://www.addgene.org/plasmid_protocols/gel_electrophoresis/ protocol] on Addgene.<br>
 +
We tried staining gels with [http://www.bio.net/bionet/mm/methods/1993-December/010023.html methylene blue] - this is a safer dye ([http://www.pro-lab.com/msds/stains_pl7027_7028_7029_methylene_blue_msds_eu.pdf MSDS]) than DNA intercalating dyes. The concentration and staining time needs a bit more adjustment to get a better signal to noise ratio. You can save the solution and re-use it many times.
 +
<br><br>
 +
 +
===== IDEXX Coliform detection =====
 +
We tested the [http://www.idexx.com/view/xhtml/en_us/water/products/colilert-18.jsf 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....<br><br>
 +
 +
'''''Results'''''
 +
 +
"incubated" a bit longer... at various temperatures for ca 40h. got positiv coliform detection at higher concentrations. also positive for e.coli in UV light.
 +
 +
[[File:IDEXX_results.jpg|320px]]
  
 +
[[File:IDEXX_results_UV.jpg|320px]]
 +
 +
[[File:IDEXX_results_UV_histo.png|320px]]
 +
<br><br><br>
 +
 +
===== Notes on PCR primer Design =====
 +
PCR primer design has great rules of thumb, but also helps to have experience and help with empirical testing. These sites are very good resources:<br>
 +
* University of Ontario, CA has nicely [http://www.molbiol-tools.ca/ organized molecular analysis links], this page specific for [http://www.molbiol-tools.ca/PCR.htm PCR primer design], including NCBI's [http://www.ncbi.nlm.nih.gov/tools/primer-blast/ primer blast page]
 +
* European Molecular Biology Laboratory (EMBL) is a great resource for nearly everything in contemporary biological research --- this is their [http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/ primer design page]
 
<br>
 
<br>
 
+
For the [http://wiki.epfl.ch/biodesign-realworld BIO-DESIGN for the REAL WORLD] project, we decided to use a primer pair designed and tested by Maheux et al. Water Research 43 (2009) pp. 3019-3028. This primer pair detects the ''tuf'' gene -and according to the paper, is quite specific for detecting ''E. coli'' and ''Shigella'':
===== Isolation of Taq expressed in ''E. coli'' =====
+
<br>
We will be comparing the pOpenTaq protocol provided by Open Biotechnology, with another protocol provided by the protein expression core facility at EPFL.
+
TEcol553 5′-TGG GAA GCG AAA ATC CTG-3′<br>
[http://hackteria.org/wiki/index.php/File:Taq_polymerase_isolation_from_E_coli.pdf Taq_polymerase_isolation_from_E_coli.pdf]
+
TEcol754 5′-CAG TAC AGG TAG ACT TCT G-3′
 +
<br><br>
 +
- more about the musings on the selection process on [http://biodesign.cc/2013/03/28/coliform-bacteria-detection-by-pcr/ biodesign.cc].
 
<br><br><br>
 
<br><br><br>
  
== Workshop Details ==
+
== Details ==
 
==== What to bring, Where to stay ====
 
==== What to bring, Where to stay ====
For the workshop itself, we will be prepare the materials as outlined above.<br>
+
For the Hack-a-Taq weekends, we will be preparing materials outlined above.<br>
 +
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'''!<br>
 +
'''Come for parts of the day, come to all the days.'''
 +
<br><br>
 
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 <br><br>
 
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 <br><br>
  
==== Dates and Times ====
+
==== Dates and Times and Meeting Points ====
 
;5 April  - Optional pre-workshop day at EPFL
 
;5 April  - Optional pre-workshop day at EPFL
: 12:15pm (EPFL [http://plan.epfl.ch/?lang=en&zoom=20&recenter_y=5864086.73818&recenter_x=730647.66058&layerNodes=fonds,batiments,labels,events_surface,events_line,events_label,information,parkings_publics,arrets_metro,evenements,evenements_informations,evenements_scenes,evenements_nourriture,evenements_boissons,evenements_entrees,evenements_presse,evenements_infirmerie&floor=3&q=SV_3615 SV3.615]) meet and work with the students of BIO-DESIGN for the REAL WORLD.
+
: 14:00pm (EPFL [http://plan.epfl.ch/?lang=en&zoom=20&recenter_y=5864086.73818&recenter_x=730647.66058&layerNodes=fonds,batiments,labels,events_surface,events_line,events_label,information,parkings_publics,arrets_metro,evenements,evenements_informations,evenements_scenes,evenements_nourriture,evenements_boissons,evenements_entrees,evenements_presse,evenements_infirmerie&floor=3&q=SV_3615 SV3.615]) meet and greet.
: 17:00 pm (EPFL) PUBLIC THESIS DEFENSE - Highthroughput  3D culture and analysis of stem cell differentiation (room and official title coming soon)
+
: 18:00 pm (EPFL [http://plan.epfl.ch/?lang=en&zoom=19&recenter_y=5864150.58636&recenter_x=730832.09484&layerNodes=fonds,batiments,labels,events_surface,events_line,events_label,information,parkings_publics,arrets_metro,evenements,evenements_informations,evenements_scenes,evenements_nourriture,evenements_boissons,evenements_entrees,evenements_presse,evenements_infirmerie&floor=5&q=BM5202 BM5202]) PUBLIC THESIS DEFENSE - Highthroughput  3D culture and analysis of stem cell differentiation (room and official title coming soon)
  
 
;6 April
 
;6 April
: 10:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market
+
: '''11:00 am''' (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market - <br>
 +
: We are running late this morning - for any questions, please contact Sachiko (at) biodesign.cc
  
 
;7 April
 
;7 April
Line 154: Line 200:
  
  
 +
'''NOW CANCELLED- 13 APRIL SESSION'''
 
;12 April  - Optional pre-workshop day at EPFL
 
;12 April  - Optional pre-workshop day at EPFL
 
: 12:15pm (EPFL [http://plan.epfl.ch/?lang=en&zoom=20&recenter_y=5864086.73818&recenter_x=730647.66058&layerNodes=fonds,batiments,labels,events_surface,events_line,events_label,information,parkings_publics,arrets_metro,evenements,evenements_informations,evenements_scenes,evenements_nourriture,evenements_boissons,evenements_entrees,evenements_presse,evenements_infirmerie&floor=3&q=SV_3615 SV3.615]) meet the students of BIO-DESIGN for the REAL WORLD.
 
: 12:15pm (EPFL [http://plan.epfl.ch/?lang=en&zoom=20&recenter_y=5864086.73818&recenter_x=730647.66058&layerNodes=fonds,batiments,labels,events_surface,events_line,events_label,information,parkings_publics,arrets_metro,evenements,evenements_informations,evenements_scenes,evenements_nourriture,evenements_boissons,evenements_entrees,evenements_presse,evenements_infirmerie&floor=3&q=SV_3615 SV3.615]) meet the students of BIO-DESIGN for the REAL WORLD.
 +
  
 
;13 April
 
;13 April
: 10:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market
+
: 10:00 am (place de la Ripponne, steps of palace de rumine) OUTFITTING THE LAB shopping in the Riponne flea market<br><br><br>
 +
 
 +
== 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 ====
 +
 
 +
[[File:GelBox_assembly.jpg|320px]]
 +
 
 +
[[File:GelBox_glueing.jpg|320px]]
 +
 
 +
 
 +
==== Testing the GelBox ====
 +
 
 +
We gave it a try using [http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-100-bp-dna-ladder-100-to-1000-bp/ GeneRuler 100bp]. To get more Voltage we connected 2 powersupplies in series. Let it run for ca 50 min and then stained with Methylene Blue 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 too far (long).
 +
 
 +
[[File:GelBox_setup.jpg|320px]]

Latest revision as of 12:21, 8 June 2014

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.
  • Lyophilized Taq mix (no cold chain needed)


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.
We tried staining gels with methylene blue - this is a safer dye (MSDS) than DNA intercalating dyes. The concentration and staining time needs a bit more adjustment to get a better signal to noise ratio. You can save the solution and re-use it many times.

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....

Results

"incubated" a bit longer... at various temperatures for ca 40h. got positiv coliform detection at higher concentrations. also positive for e.coli in UV light.

IDEXX results.jpg

IDEXX results UV.jpg

IDEXX results UV histo.png


Notes on PCR primer Design

PCR primer design has great rules of thumb, but also helps to have experience and help with empirical testing. These sites are very good resources:


For the BIO-DESIGN for the REAL WORLD project, we decided to use a primer pair designed and tested by Maheux et al. Water Research 43 (2009) pp. 3019-3028. This primer pair detects the tuf gene -and according to the paper, is quite specific for detecting E. coli and Shigella:
TEcol553 5′-TGG GAA GCG AAA ATC CTG-3′
TEcol754 5′-CAG TAC AGG TAG ACT TCT G-3′

- more about the musings on the selection process on biodesign.cc.


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)


NOW CANCELLED- 13 APRIL SESSION

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 Methylene Blue 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 too far (long).

GelBox setup.jpg