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DIYbio Toronto Message Board › PCR Horsemeat Detection

PCR Horsemeat Detection

Andrew
user 10735347
Toronto, ON
Post #: 9
Do we need to change the next Equipment Jam to a Reagent Jam?
Michael Y.
user 94483532
Shrewsbury, MA
Post #: 1
As for buying reagents:

For this sort of experiment you just need the lowest grade of DNA polymerase since you're not after product accuracy but just product size. Taq is just fine; using better stuff like ipfu/Phusion/Q5 is a waste of money.

As for saving money of course it's often cheaper to just mix the master mixes by yourself. I've personally never used master-mix.

Nitrile gloves are a must, if it's not necessary latex can do. Your hands are more dangerous to PCR than PCR is to your hands. Bits of skin cells falling in might mean that DNAses just got into the reaction mix :P
Ewan
user 12048956
Toronto, ON
Post #: 19
Yeah, the Taq-based froggamix is only $10, take a look at the link from the spreadsheet. Can't go wrong there!

On balance, the individual ingredients for the various solutions like the lysis buffer are really quite pricey, so I think we're best to go with a DNA extraction kit. I've added links.

I went to the medstore today and they said that we should be OK to buy from them. I've got to chat with a higher level supervisor to confirm though, but it looks good.

Can anyone help fill in what we need for electrophoresis? I'm a bit lost there. Once that's done, we're actually ready to roll. Alaina will be able to take delivery of primers, as she has a commercial location, and the kits don't require heavy-duty cold storage, according to their manufacturers.
Ewan
user 12048956
Toronto, ON
Post #: 20
Here's a link to the manual for Duane's machine. Let's start reading and get nice and familiar with it:
http://www3.appliedbi...­
Michael Y.
user 94483532
Shrewsbury, MA
Post #: 4
Reagent list for electrophoresis:

- Distilled water (minimum, preferably use double-distilled water (ddH2O) or Milli-Q water, which is a step up from ddH2O)
- Ethidium Bromide solution, in water (Duane's stuff seems really old...)
- Biotech grade Agarose (NOT Agar...) - this stuff is about $200 for a 500g bottle, but that's enough for 500 gels. It's expensive lump-sum but not so bad on a per-use basis

I assume you have all the equipment you need...?
Ewan
user 12048956
Toronto, ON
Post #: 21
Here's a link to the manual for Duane's machine. Let's start reading and get nice and familiar with it:
http://www3.appliedbi...­
Samantha C.
the_io
North York, ON
Post #: 18

- Ethidium Bromide solution, in water (Duane's stuff seems really old...)
Did we vote against EtBr alternatives?
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