DIYbio Toronto Message Board › PCR Horsemeat Detection

PCR Horsemeat Detection

Ewan
user 12048956
Toronto, ON
Post #: 8
Let's get our PCR on!

I gather we'll need:

  • Mastermix (dNTP, buffer, Taq polymerase)
  • Primers
  • Electrophoresis equipment
  • An OpenPCR machine

If you know we need anything else at all (like pipettes, which I imagine we will) and/or have other crucial information that we've missed, please do chime in.

I'm for buying primers from a supplier without having to design them, so we can focus on the core process. I've been a bit confused by the ones I can find (linked below), so if anyone can enlighten us, that'd be lovely.

Some of the commercial mixes/primers are designed for qPCR. Is it possible to skip electrophoresis if we use them, and just coarsely measure whether fluorescence has passed a minimal threshold, or am I completely out to lunch?

Finally, what do we need for electrophoresis? It looks like we just need a very simple machine, some volts, and some gel.

Links:

PCR machine and mastermix (Thanks Samantha!):
http://www.openbiotec...­

Meat speciation PCR primer/kit supplies:
http://www.neogen.com...­
http://www.invitrogen...­
http://www.primerdesi...­

Electrophoresis:
http://www.iorodeo.co...­
http://openwetware.or...­
http://citizenscience...­
http://danielpacker.o...­
Christopher
user 82448352
Toronto, ON
Post #: 3
This looks like a great list Ewan!

The qPCR mixes, based on my understanding, are measured by fluorescence, and as you alluded to, do not require electrophoresis. The reagents though are expensive, and need specifically designed reporter probes that are complementary to your DNA sequence of choice. One way to minimize costs and improve flexibility is to use more generic double stranded DNA binding dyes (I think we use SYBR Green in our lab). These will fluoresce any DNA that is double stranded, which should theoretically only be the product of the PCR. However "primer dimers" are a common false reading, but it should be ok if we're only looking for coarse fluorescence readings.

Electrophoresis generally only requires what you said:

a) A simple machine that is essentially a large tank that is designed to hold the gel and pass a controlled electric current from one end to the other

b) A gel, and of course everything required to make a gel. I wont list everything here, but most gel kits will come with the required materials (Agarose for DNA, Polyacrylamide for Proteins, gel casings, buffer, etc...)

c) We need a DNA ladder. This is an engineered mix of DNA strands, at preset basepair lengths of various sizes. Its a sample you run as a control in your gel, and it gives an idea of where certain DNA sizes with migrate to (remember a gel is designed to separate the DNA strands by size)

d) We need loading dye. DNA is invisible, and loading the gel is problematic if you cant see if your sample made it in or not. Loading dyes are generally a mixture of Bromophenol blue and other compounds based on what you're sampling (we can look up specific recipes later). This also changes the density of the sample, so they easily sink into the wells; otherwise, the sample will just disperse and never set in.

e) Micropippettes

f) Finally we need something to visualize the DNA after the gel is complete. Ethidium Bromide is a natural stain which intercalates between the DNA molecules. However it only fluoresces under UV light, which means we'll need some source of that to visualize results.

All in all, its probably much simpler to use qPCR reagents at the beginning, since the capital required for electrophoresis is pretty steep. However the qPCR reagent price difference will eventually make this the more expensive choice
Doug H
user 85970252
Toronto, ON
Post #: 1
Thanks for the research and extra info Ewan and Christopher.

I'm really interested in doing some of these experiments and am happy to put up some money to that end.

We can get really cheap UV light sources at A1 parts or active surplus. Also the electrophoresis equipment looks like it could be hacked up easily too.

Out of curiosity, would this equipment work for DNA barcoding experiments too?

https://en.wikipedia....­
http://www.dnabarcodi...­
Ewan
user 12048956
Toronto, ON
Post #: 9
Well, it looks like those primers are way too expensive, so we'll have to start cold and design our own.

I am utterly at a loss as regards how we'd do this, so can anyone get us through this part? It looks like this is the hardest piece. I couldn't find any information about what sequences are used by the primers we looked at, and don't even know where to start finding out for myself.
roberta b.
user 39448542
Toronto, ON
Post #: 17
how to make cheap gel
http://learn.genetics...­
Andrew
user 10735347
Toronto, ON
Post #: 1
Hi Ewan,

I've never tried it with DNA primers, but I know that with other industries you can often call around to a few suppliers, explain who you are and what you're trying to do, and end up with free or very cheap samples. They're often very helpful with tips and ideas, too. If they can't help you, you always end the call with, "Do you know anybody else I could try to get in touch with to help me with this?"

Some places will brush you off, and some places will be super helpful and point you in the direction of things you never even thought of.
Ewan
user 12048956
Toronto, ON
Post #: 13
Update:
It looks like Naveen and Chris will be able to get us custom primers made at a nice low price. The pre-made stuff was a dead end due to cost.

That's the last piece of the puzzle, so we're finally ready to order an OpenPCR machine. We'll need to get an Electrophoresis box as well.

This page is really helpful for sequences and techniques:
http://openwetware.or...­

I say we drop $130 on this electrophoresis box:
http://www.iorodeo.co...­

I'm going to double check we have everything we need to start, and then start contacting people to collect pledges and get this puppy ordered. Please PM me or reply to this thread if you'd like to chip in. The more the merrier!
Andrew
user 10735347
Toronto, ON
Post #: 2
I'm in for $20 (or $30, if we need a top-up).
Andrew
user 10735347
Toronto, ON
Post #: 8
From what I'm reading, the DNA extraction from meat will be more challenging than what we've done in the past with fruit. Here are a couple of protocols for DNA extraction from muscle:

http://www.foodbase.o...­

http://www.sfu.ca/bis...­

http://www.researchga...­

http://current-biolog...­

http://www.bio.net/mm...­

One thing that most of them mention is proteinase K. How hard would it be to get our hands on some of that?
Ewan
user 12048956
Toronto, ON
Post #: 18
Yeah, I just reached that same conclusion and had been looking through The Simple Fool's Guide to PCR (herafter referred to as SFGP), which makes extraction look quite easy:
http://palumbi.stanfo...­

I've been poking around www.uoftmedstore.com to try and price things out and see if they're available. Shipping and relationships with suppliers are proving *very* difficult at this stage. With that in mind, I think we need to try and stick to them for now and hope we can get our supplies through them. We don't even need the openbiotech mastermix, as we can buy FroggaMix from the medstore as well.

Here are my thoughts on the extraction protocols we have:

SFGP: Looks like the easiest of the lot. They don't actually mention adding Proteinase K or incubating, though they mention it as an ingredient to be added to the Lysis buffer in an appendix. I wonder if Proteinase K needs incubating or not? Since it breaks down all that protein, I assume it really helps to add it. It's not expensive on the medstore.

Foodbase: These all seem to use proprietary components, which are terrifyingly pricey. We probably don't need anything this good for a first run, especially given that these are for RT-PCR designed for determining quantity present. That said, they look very similar to the SFGP protocols on a cursory read. It may make sense to graduate to these once we can afford to, and the medstore does sell wizard. Good find, that was an interesting read!

SFU: This seems good, but requires an incubator. It seems highly purity focussed, which is probably less important given that we're on the plain-PCR train.

Researchgate: Commercial kits again. UoT Medstore lists DNEasy at $143 for 50 preps, which is where wizard products *start* (I'm still not sure which we'd want). Interesting to note how food products are difficult because of so many things that may inhibit PCR, hence the importance of good extraction. I still think we can start with plain meat and see what happens. We can always buy Wizard or DNEasy if we have a problem.

Current-biology: DNEasy again

Bio.net: This looks simple and cheap. Interesting step to use the freeze-and-grind technique.

In conclusion, I think we should try the SFGP and Bio.net methods. The others require most of the same ingredients, so it makes sense to try the simplest first and see how it goes. Given how complicated this step seems to be, it wouldn't hurt to do this experiment alone before the PCR itself, just to see how we do.

I've put up a project spreadhseet with things we'll need for each stage, purchase links which are only from the medstore, and storage requirements. Please do help fill it in!
https://docs.google.c...­

As usual, comments from anybody who actually knows what they're doing are greatly appeciated.
Powered by mvnForum

Our Sponsors

  • Synbiota Inc.

    Web-based DIYBio Development, Collaboration, & Wetware Kits

People in this
Meetup are also in:

Sign up

Meetup members, Log in

By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy