Difference between revisions of "BioLab Manual"

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= Safety =
 
= Safety =
−
Please be aware that the substances you are working with maybe highly toxic. Consult [[Substance and Toxicity databases]] before if you are unfamiliar with the compounds with which you will be working.
 
  
−
=== No mouth pipetting ===
+
All users of the Biolab must follow the [[BioLab Usage Guidelines]] for their own and other user's safety.
  
−
This is fairly self explanatory, and is also specifically mentioned in the regulations. (I presume due to some fairly horrific accidents)
+
= Protocols =
−
 
 
−
=== No unlabelled containers ===
 
−
 
 
−
Unlabelled containers are a hazard, even if they are not hazardous.
 
  
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It makes it difficult to keep the lab tidy if there are random concoctions cluttering the shelves.
+
Instructions for [[E-coli RFP Transform |(a "Blink LED" for easy first transformations)]]
  
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Please label with the following;
+
Instructions for [[Stock Solutions|making general stock solutions]]
  
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* nick
+
For more extensive databases of protocols:
−
* date
+
* http://www.protocolpedia.com/index.php
−
* contents
+
* http://openwetware.org/wiki/Protocols
  
−
and if you are going to be leaving something hazardous or plan to leave an experiment running for an extended duration, please mail the list.
+
== Established Protocols ==
−
 
 
−
= Protocols =
 
−
 
 
−
== Established ==
 
 
The protocols we have got working in the Biohackspace:
 
The protocols we have got working in the Biohackspace:
  
 
* [[DNA extraction using Chelex 100]]
 
* [[DNA extraction using Chelex 100]]
 
* [[DNA extraction and precipitation with ethanol / isopropanol]]
 
* [[DNA extraction and precipitation with ethanol / isopropanol]]
 +
* [[DNA extraction and precipitation with phenol-chloroform]]
 
* [[Make and run an agarose gel]]
 
* [[Make and run an agarose gel]]
−
* Growing Escherichia coli
+
* [[Solid Media Bacterial Culture]]
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* Growing Vibrio fischeri ([[Project: Growing bacteria|our first attempt]])
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* [[Liquid Media Bacterial Culture]]
 +
* [[PCR]]
 +
* [[Miniprep | Anachem Miniprep (plasmid purification)]]
 +
* [[LBA]] (LB ampicillin)
  
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== Untested ==
+
We also have [https://github.com/london-biohackers/biohackspace-sops/blob/master/index.rst protocols on our github SOPs page]
  
−
The protocols we we would like to get working in the Biohackspace.
+
== Untested Protocols==
 +
The protocols we would like to get working in the Biohackspace.
  
−
* [[E. coli GFP transformation]]
+
== Media Recipes ==
−
 
 
−
== Media ==
 
  
 +
* [[LB Medium]]
 +
* [[BOSS Medium]]
 +
* [[HS Medium]]
 +
* [[Kombucha Medium]]
 +
* [[TBE]]
  
 
= Equipment =
 
= Equipment =
Line 65: Line 64:
  
 
== Strains ==
 
== Strains ==
−
* [[E. coli DH5α]]
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* [[E. coli DH5α|''E. coli'' DH5α]]
−
* [[V. fischeri]]
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* ''[[V. fischeri]]''
−
* [[P. polycephalum]]
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* ''[[P. polycephalum]]''
 +
* ''[[G. hansenii]]''
 +
* ''[[Dinoflagellate|''P. fusiformis, P. lunula'']]
  
 
== Reagents ==
 
== Reagents ==
−
The reagents we currently have stocked:
+
[[:Category:BiolabInventory]]
  
−
* A little bit of Monica in my life
+
The reagents we currently have stocked
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* A little bit of Erica by my side
 
−
* A little bit of Rita is all I need
 
−
* A little bit of Tina is what I see
 
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* A little bit of Sandra in the sun
 
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* A little bit of Mary all night long
 
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* A little bit of Jessica here I am
 
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* A little bit of you makes me your man
 
  
 +
{| class="wikitable sortable" style="float:left"
 +
|+ General Reagents
 +
! Reagent !! Brand !! Location
 +
|-
 +
|Bacto Agar || || Shelves
 +
|-
 +
|Bacto Yeast Extract || || Shelves
 +
|-
 +
|LB broth powder || || Shelves
 +
|-
 +
|TBE buffer x10 || || Shelves
 +
|-
 +
|Sodium Acetate || || Shelves
 +
|-
 +
|Ethidium Bromide || || Drawers
 +
|-
 +
|Tri-Sodium Citrate-2 Water || || Shelves
 +
|-
 +
|Magnesium Chloride || || Shelves
 +
|-
 +
|Calcium Chloride || || Shelves
 +
|-
 +
|Agarose || || Shelves
 +
|-
 +
|Glycerin || || Shelves
 +
|-
 +
|Ampicillin Sodium Salt (powdered) || || Fridge
 +
|-
 +
|Kanamycin Sulphate (powdered) || || Fridge
 +
|-
 +
|IPTG || || Freezer
 +
|-
 +
|Proteinase K 1mg/ml || || Freezer
 +
|-
 +
|}
  
−
    link to shopping list G spreadsheet
+
{| class="wikitable sortable" style="float:left"
 +
|+ DNA (Primers, Plasmids, etc.)
 +
! Reagent !! Description !! Brand
 +
|-
 +
| [http://en.wikipedia.org/wiki/PUC19 pUC19] control DNA || [https://www.neb.com/products/n3041-puc19-vector NEB #N3041A] || NEB
 +
|-
 +
| [https://www.neb.com/products/m0491-q5-high-fidelity-dna-polymerase Q5 Hi-GC enhancer] || 5x concentrate || NEB
 +
|-
 +
|}
  
 +
{| class="wikitable sortable" style="float:left"
 +
|+ Restriction Enzymes
 +
! Reagent !! Cutting Site !! Brand
 +
|-
 +
|}
 +
<div style="clear:both;"></div>
  
 
See also: [[Project:Making our own reagents]]
 
See also: [[Project:Making our own reagents]]
  
  
 +
 +
    link to shopping list G spreadsheet
  
 
== Suppliers ==
 
== Suppliers ==
Line 108: Line 153:
  
 
Also see general hackspace [[suppliers]] page.
 
Also see general hackspace [[suppliers]] page.
−
 
−
=== DNA extraction===
 
−
 
−
* Triton X-100
 
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*Triton + TBE + meat tenderiser solution.
 
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*Proteinase K solution (We think it's 10mg/ml but not 100% sure)
 
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*Chelex 100 sodium form. (25g) from [http://www.sigmaaldrich.com/catalog/product/SIGMA/C7901?lang=en&region=GB sigma] for £55.08. We also have some of this suspended in tap water
 
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*70% isopropanol (rubbing alcohol) - widely and cheaply available
 
−
 
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===PCR and restriction digest===
 
−
 
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*[https://www.sigmaaldrich.com/catalog/product/sigma/p2893?lang=en&region=GB Taq ReadyMix]. Click on bulletin for useful info about procedure for using it. £144 + £30 shipping for 100 reactions from [http://www.sigmaaldrich.com/catalog/product/sigma/p2893?lang=en&region=GB sigma]. We can halve reaction volume with no adverse effects giving us 200 reactions.
 
−
* [http://www.sigmaaldrich.com/catalog/product/sigma/s4438?lang=en&region=GB SYBR green taq readymix] £115.92 for 200 reactions
 
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*dH20 (deionised water bought from garages).
 
−
 
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* [https://lists.kentgeek.org/pipermail/lhs-biohacking/2012-January/000210.html Post] on reconstituting primers
 
−
 
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'''Amelogenin'''
 
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Gene associated with tooth mineralisation on human X and Y chromosomes.
 
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Sequences: 5'-CTGATGGTTGGCCTCAAGCCTGTG-3' and 5'-TAAAGAGATTCATTAACTTGACTG-3'
 
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Produces product 977 bp long (human X chromosome) or 788 bp long (human Y chromosome).
 
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'''Amelogenin'''
 
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Gene associated with tooth mineralisation on human X and Y chromosomes.
 
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Sequences: 5'-CCCTGGGCTCTGTAAAGAATAGTG-3' and 5'-ATCAGAGCTTAAACTGGGAAGCTG-3'
 
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Produces product 106 bp long (human X chromosome) or 112 bp long (human Y chromosome).
 
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Anneal at: 66 C (source: http://biowww.net/gene/gene-AMELY.html)
 
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'''SRY'''
 
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Sex-determining region Y.
 
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Sequences: 5'-ATAAGTATCGACCTCGTCGGAAG-3' and 5'-GCACTTCGCTGCAGAGTACCGAAG-3'
 
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Produces product 93 bp long (human Y chromosome).
 
−
 
−
* KPN1 (£27 for 1000 units from [http://www.sigmaaldrich.com/catalog/product/sigma/r1258?lang=en&region=GB sigma] - a unit is "the enzyme activity that completely cleaves 1 µg λ DNA in 1 hr. at 37 °C in a total volume of 25 µl of Buffer SL for restriction enzymes.". Quantity not listed but I estimate 115ul) and ALU1 (£18.90 for 100 units from [http://www.sigmaaldrich.com/catalog/product/sigma/r6885?lang=en&region=GB sigma] - can also buy 500 units for £33.80) for blood typing. I guess we will be using it on minimum 20ng of DNA, which gives us 50 reactions for Kpn1 and 5/25 reactions for Alu1.
 
−
 
−
===Electrophoresis===
 
−
 
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* "SYBR". Small vial. Stain. Supplier: bugs
 
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* SafeWhite nucleic acid stain. NBS Biologicals, cat NBS-SW1
 
−
* DNA Marker-D (100 bp-2000 bp). This is the "ladder" for electrophoresis comparisons. Supplied by NBS Biologicals, nbsbio.co.uk, cat: GM335
 
−
*1KB DNA ladder. Current ladder is aliquoted with loading dye from a 50 ug sample of concentration 0.2 ug / ul. [http://www.taq-dna.com/rich_files/attachments/DNA_Ladders_DNA_Weight_markers_DNA_leiter/1000_bp_1kb_DNA_Ladder.pdf key]. £36 + £15 shipping for 50ug (120 uses) from [http://www.nbsbio.co.uk/search.asp?s=ladder&Submit.x=0&Submit.y=0 nbsbio]
 
−
*Ethidium bromide stock solution - 2 ul ethidium bromide solution to 40 ml water.
 
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*Powdered agarose (£46.80 + £15 shipping for 100g. Enough for 200 gels) from [http://www.nbsbio.co.uk/product.asp?pID=12205&cID=316 nbsbio].
 
−
*[http://www.nbsbio.co.uk/product.asp?pID=6241&cID=75 Safewhite 1ml]
 
−
*[http://www.nbsbio.co.uk/product.asp?pID=10571&cID=33 DNA Ladder]
 
−
*10x TBE - £33.60 for enough powder for 4L (enough for 267 50ml 1% TBE gels + 100ml 1% TBE buffer) from [http://www.nbsbio.co.uk/product.asp?pID=12335&cID=321 nbsbio]
 
−
*[http://wiki.london.hackspace.org.uk/view/Project:Biohacking/Stains List of possible DNA stains]
 
  
 
[[Category:Biohacking]]
 
[[Category:Biohacking]]

Latest revision as of 19:58, 2 October 2016

Safety

All users of the Biolab must follow the BioLab Usage Guidelines for their own and other user's safety.

Protocols

Instructions for (a "Blink LED" for easy first transformations)

Instructions for making general stock solutions

For more extensive databases of protocols:

Established Protocols

The protocols we have got working in the Biohackspace:

We also have protocols on our github SOPs page

Untested Protocols

The protocols we would like to get working in the Biohackspace.

Media Recipes

Equipment

To create a page for a piece of lab equipment fill out this form and make sure you put "Biohacking" as the subcategory.

We also have an array of glassware and consumables.

This site has a directory of service engineers for various biomedical/biochemical equipment:

http://www.labwrench.com/?equipment.requestAQuote/categoryNo/707/Mass-Spectrometers/

Strains

Reagents

Category:BiolabInventory

The reagents we currently have stocked

General Reagents
Reagent Brand Location
Bacto Agar Shelves
Bacto Yeast Extract Shelves
LB broth powder Shelves
TBE buffer x10 Shelves
Sodium Acetate Shelves
Ethidium Bromide Drawers
Tri-Sodium Citrate-2 Water Shelves
Magnesium Chloride Shelves
Calcium Chloride Shelves
Agarose Shelves
Glycerin Shelves
Ampicillin Sodium Salt (powdered) Fridge
Kanamycin Sulphate (powdered) Fridge
IPTG Freezer
Proteinase K 1mg/ml Freezer
DNA (Primers, Plasmids, etc.)
Reagent Description Brand
pUC19 control DNA NEB #N3041A NEB
Q5 Hi-GC enhancer 5x concentrate NEB
Restriction Enzymes
Reagent Cutting Site Brand

See also: Project:Making our own reagents 


   link to shopping list G spreadsheet

Suppliers

  • Sigma-Aldrich - Good for custom primers
  • Cole-Parmer
  • Farnell
  • Chang bioscience - pipettes
  • NBSBio - Good for ladders and cheap agarose (only low concentration) NB: Be sure to navigate to http://www.nbsbio.co.uk NOT http://nbsbio.co.uk, or their payment system doesn't work. Weird. Standard shipping cost is £15
  • Web scientific - Taq and ladder
  • New England Biolabs They sell very cheap taq. We could also look at their restriction enzymes. However atm they won't sell to us as they only sell to universities or registered biolabs. See this post and this post. Once we have all our registrations for LBL sorted we could try again.
  • Thermoscientific - Recommended for good value restriction enzymes. Not tried yet.
  • VWR - Big supplier of chemicals, glassware and various lab stuff. Sounds very positive about selling to the hackspace.
  • Invitrogen We failed their due diligence check. See post here and here. They have cheap TBE.
  • IDT - possible source for primers. Haven't tried them yet.
  • cheaplabstuff (ebay) - Cheap 1.5ml and 15ml tubes in small quantities. Also pasteur pipettes.
  • microsupplies (ebay) - Cheap 5-200ul and 100-1000ul tips by the 1000
  • Cambridge Bioscience - Not yet used, may supply GelRed or GelGreen (enquiry by Bene)

Also see general hackspace suppliers page.

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