Group:Biohacking: Difference between revisions

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*November/December 2012 - Refine DNA extraction process to get it working consistently, and complete blood typing and sex typing experiments. Also do the self cloning protocol from NCBE to learn transformation procedures.
*November/December 2012 - Refine DNA extraction process to get it working consistently, and complete blood typing and sex typing experiments. Also do the self cloning protocol from NCBE to learn transformation procedures.
*November 2012 - early 2013 - Get a larger lab space with a licence to do genetic modification, and get the funding to do this.
*November 2012 - early 2013 - Get a larger lab space with a licence to do genetic modification, and get the funding to do this.
Wellcome Trust grant 
Of  the collaborative projects that were agreed upon in Paris the most serious one one involved making our own  restriction enzymes and taq. polymerase.  The purpose of this would be to create an alternative source of supply for the DIY bio movement, one  that is independent of the commercial suppliers that we currently use. The problem with this is, that we might not be able to compete with the commercial suppliers in terms of cost, simply because we cannot apply the economies of scale that they can - we would be going back to artisanal, rather than mass, production. A way round this might be to produce a public biobrick with the taq gene inserted, which would then be usable over and over again, thus reducing the cost of production. Or perhaps such a biobrick already exists? If so, we could  use it. This involves getting a bacterium to express taq, restriction enzymes etc.(not at the same time of course!) Our last collaborative project involve inserting the gene for mercuric reductase into e coli, so in principle at least, we have done this before. The expressed  enzymes  must not however interfere with the bacterium's own DNA so the technical problems are quite considerable.  We would need a license to do genetic engineering. Hopefully we would be able to get in the new space.


== People ==
== People ==

Revision as of 20:31, 14 January 2013

Biohacking / DIYBio at LHS

We are the biohacking group at the London Hackspace, a mix of amateur and professional biologists, attracted by the potential of DIYbio and synthetic biology. Anyone is welcome, so get involved! It's fascinating, the field and community is growing all the time, together with the ability of amateurs to do cool stuff. Over the past year we've been developing our equipment, optimising techniques and running some research projects. Here's quite a good short local news video showing our lab and some of what we do in it.

Our current projects / schedule

We're currently working on genetic testing, identifying specific genes using PCR and electrophoresis. We've been working mainly on sex typing with amelogenin, and have just started working on plant species testing to refine our DNA extraction and PCR process. In August and September we worked with the UCL igem team to develop a 'public biobrick'.

Our big goal for the next year is to begin projects involving genetic modification. For this we'll need to become certified as a class 1 lab, we are currently collecting information on how to do this.

Schedule

  • November/December 2012 - Refine DNA extraction process to get it working consistently, and complete blood typing and sex typing experiments. Also do the self cloning protocol from NCBE to learn transformation procedures.
  • November 2012 - early 2013 - Get a larger lab space with a licence to do genetic modification, and get the funding to do this.
Wellcome Trust grant  
Of  the collaborative projects that were agreed upon in Paris the most serious one one involved making our own  restriction enzymes and taq. polymerase.  The purpose of this would be to create an alternative source of supply for the DIY bio movement, one  that is independent of the commercial suppliers that we currently use. The problem with this is, that we might not be able to compete with the commercial suppliers in terms of cost, simply because we cannot apply the economies of scale that they can - we would be going back to artisanal, rather than mass, production. A way round this might be to produce a public biobrick with the taq gene inserted, which would then be usable over and over again, thus reducing the cost of production. Or perhaps such a biobrick already exists? If so, we could  use it. This involves getting a bacterium to express taq, restriction enzymes etc.(not at the same time of course!) Our last collaborative project involve inserting the gene for mercuric reductase into e coli, so in principle at least, we have done this before. The expressed  enzymes  must not however interfere with the bacterium's own DNA so the technical problems are quite considerable.  We would need a license to do genetic engineering. Hopefully we would be able to get in the new space.

People

Andy, Joel, Mike, Nicholas, Paddy, Paul, Simon, Taylor, Tom, Tonderai, Will

How to find us

  1. Come to a meeting: we meet at 7pm every Wednesday in the London Hackspace: Unit 24, Cremer Business Centre, 37 Cremer Street, London E2 8HD. Email Nicholas if you can't find the place.
  2. Post on the biohacking mailing list. You can view the mailing list archives through the previous link or through the google groups interface.

Membership

We encourage you to become a member of the biohacking group. Biohacking is more expensive than the typical hackspace activity, and with your membership we can pay for chemicals, primers, and any random equipment we may need.

To become a member, set up a standing order to the London Hackspace with the reference "biohacking". You need to become a hackspace member before you can see the direct debit details. Look here for more information. If you want to become a Biohacking member without joining the space (you really should join the hackspace! It's great), get in contact with Nicholas.

The suggested donation is £2 a week.

Our standard protocols / how to's

Our reagents + equipment

  • Thermal Cycler (PCR Machine) Perkin Elmer 480
  • Gel electrophoresis box
  • Incubator with shaker
  • Two centrifuges - estimated speed 5000 rpm
  • Wet stuff: our inventory of primers, buffers, stains...
  • Various flasks and beakers
  • Two scales (one down to 0.1g, the other down to 0.01g)
  • 1-10ul pipette + tips. Tips available for £27.60 from NBSbio
  • 5-50ul pipette + tips
  • 100-1000ul pipette
  • Pasteur pipettes
  • 0.5ml PCR tubes. 1000 for £24 from nbsbio
  • 1.5ml tubes. 100 for £3 from ebay
  • Pressure cooker (goes up to 15PSI and 121°C (this is industry standard, not actually tested) to be used as autoclave. Autoclave tape.

Shopping list in priority order (See wet stuff for sources and prices not listed here)

These prices are the first I found. We should look for better ones.

  • Agarose (run out)
  • Ladder (only a few more gels left for older ladder)
  • 5-200ul tips (one box left)
  • KPN1 (£27 for 1000 units from 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.") and ALU1 (£18.90 for 100 units from 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.
  • 0.65ml tubes (or whatever size will fit in PCR machine) (around 20 tubes left)
  • Some 1.5ml tubes for reactions of higher volume (such as ethanol precipitation). Don't need too many
  • Taq readymix (less than 20 reactions left of SYBR green taq mix)
  • 10X TBE (running very low).
  • Jim paid £108.99 for company formation + registered office etc. Needs to be reimbursed.
  • 15ml tubes
  • 1-10ul tips (a few left but running low)
  • 100-1000ul pipette tips for new pipette. This ebay seller sells 1000 for £8.90 + shipping


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.
  • 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 http://eu.idtdna.com/site?c=UK] - possible source for primers. Haven't tried them yet.
  • cheaplabstuff (ebay) - Cheap 1.5ml and 15ml tubes in small quantities
  • microsupplies (ebay) - Cheap 5-200ul and 100-1000ul tips by the 1000

Also see general hackspace suppliers page.

Wish list

  • Autoclave strips. These supposedly tell you if the autoclaving has been done at the required conditions, unlike autoclave tape which only tells you if the temperature has been reached.
  • If extractions keep failing, lets look at some extraction kits. This one recommended by Tom is ∼£100 for 100 reactions or ∼£250 for 500 extractions.
  • A more consistent and effective way of crushing/homogenising small amounts of tissue for DNA extraction. Blenders are good but not for small quantities. Pestles are ok, but not great. One idea is to make a small pestle by melting a pipette tip then using a PCR tube as a mortar. Some labs use liquid nitrogen or a sonicator.
  • Spectrophotometer for measuring DNA concentration. Simon and Tom are on this.
  • Another centrifuge, if ours breaks down, or a dremelfuge. One option is a Dremelfuge - microcentrifuge to be made on a 3D printer. Can print a new one, or buy pre printed for £50 from shapeways. Another option could be this £100 13,000rpm centrifuge from ncbe. Says it's only for schools but we can try and persuade them. If we want a chilled centrifuge we can put it in the fridge / freezer.
  • Another PCR machine. One option is the Open Source, Hackable PCR machine. Another is the Light Bulb PCR less than $50 to build this machine (including the $30 arduino)

Costs (assuming prices on wet stuff)

Per Chelex DNA extraction:

  • Chelex: £0.33 (0.15g)
  • Pasteur pipettes: Not calculated
  • Tubes: £0.04 (1 for chelex incubation, 1 to save supernatant)
  • Tips: £0.06 (1 for mixing chelex, 1 removing supernatant)

Total per sample: £0.43 + pasteur pipettes

Per PCR reaction Assume 25ul total volume. 12.5ul Taq, 5ul primers

  • Taq: £0.72 (12.5ul)
  • Primers: Not calculated but likely to be small
  • Tubes: £0.02 (1 tube for PCR)
  • Tips: £0.15 (Assuming 1 tip for template, 1 for PF, 1 for PR, 1 for dH20, 1 for Taq)
  • Paraffin: Negligible

Total per sample: £0.89 + primers

Per restriction digest for blood typing: This estimate may be wrong. Need to calculate quantities definitively

  • KPN1 and ALU1: £2.08

Total per sample: £2.08 + another gel

Per sample on a gel: For each sample:

  • Tubes: £0.02 (1 to mix loading buffer with PCR product)
  • Tips: £0.03 (1 to add product to loading buffer and then load into well)
  • Loading buffer: Negligible

Total per sample: £0.05

Per gel: Assume a 1% 50ml agarose gel with 100ml buffer and 2.2ul EtBr

  • Agarose: £0.23
  • TBE: £0.60
  • Ladder: £0.30
  • Tips: £0.03 (1 to add loading buffer to all tubes)
  • dH20: Negligible
  • EtBr: Negligible

Total per gel: £1.16

So for a chelex extraction, PCR and gel it costs at least £1.37 per sample. A gel costs at least £1.16, so a typical session involving 4 extractions + PCRs, followed by a gel costs at least £6.61.

Ideas for cost+efficiency savings

  • Cheaper Taq would make the biggest contribution. Investigate NEB (won't sell to us at the moment), web scientific and nbs.
  • Smaller gels and gel box could halve amount of TBE and agarose used.
  • Reduce concentration of agarose and TBE in gel and buffer. E.g. we could use 0.5% TBE not 1%.
  • Use TAE instead of TBE (TAE is 1/2 to 1/3 the price of TBE). However it is claimed TBE is better for small DNA lengths like ours
  • Buy powdered TBE. NBSBio - £33.60 for 1 litre of 10x (50p per gel+buffer), and £76.80 for 5L (23p per gel+buffer). Dry premix powders are even better value than this. £18 for 1L (27p per gel+buffer), and £28 for 4L (13p per gel+buffer).
  • Make our own TBE. Recipe here. Advantage that we could also make TE.
  • Invitrogen do http://products.invitrogen.com/ivgn/product/15581044# cheaper TBE] but won't sell to us atm
  • Cheaper sources for tubes and tips e.g. ebay
  • Heated lid making paraffin unnecessary. Save on paraffin + extra tubes for mixing loading buffer with PCR product as loading buffer could be added directly to it if there are no further plans for it. Mostly efficiency saving rather than cost.
  • Same result could be achieved by using a taq readymix with loading buffer included (e.g. sold by webscientific)
  • Larger quantities of agarose. Our agarose from NBSBio works out at £0.23 per gel for 100g, but if we bought 500g it would be £0.18 per gel. Not a massive saving though.

Resources

Education

A primer for synthetic biology

Community

PCR primer design

Proposed code of conduct

See here: http://wiki.hackspace.org.uk/wiki/Biohacking/Code_of_Conduct

FAQ about DIYBio

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