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Paper disk DNA preparation

After watching too much CSI I saw that genomic DNA was surprisingly stable at room temperature and could be isolated from an array of juices using cotton buds etc. It's kind of like making pressed flowers or a herbarium.

As there is already an excellent instuctable for a PCR machine (http://www.instructables.com/id/Coffee-Cup-PCR-Thermocycler-costing-under-350/ ), a method for rapid DNA template isolation may be of use.

SAFETY - Most people or animal juices have the potential for the transmission of pathogens so please take appropriate precautions.

Never lick the spoon when you are doing (non food based) Science!

I have used this method thousands of times with varrious plant materials. For Arabidopsis thaliana  and Medicago truncatula  the filter paper worked well, for more crunchy leaves from plants such as Ricinus communis I could get a green juice out but not much luck with PCR products, it could be the more robust leaf architecture prevented genomic DNA leaking out as well or it could have been inhibitory factors.

Have fun.


Materials

-Non absorbent disposable backing
    I used parafilm (like a thick clingfilm) but clingfilm (e.g. saran wrap) could be used.

-Absorbent paper
    I used filter paper. Tissue paper is proably too thin but a thin absorbent craft card may work.

-Pounding implement
    I used a plastic container, you need to crush the leaf so anything from a hammer to a plastic cola bottle could be used.

-Hole punch
    I had access to medical biopsy punches and I could get a good few uses before it got too blunt. A standard ring-binder hole punch will give you disks but they will be big and soak up a lot of PCR mix. Leather punches for belt holes work a treat.

Step 1: PCR basics

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An excellent introduction to PCR can be found on Wikipedia here .

Briefly PCR (polymrease chain reaction) is a method by which a small number of copies of a specific sequence can be amplified many thousands of times. This would be useful to generate enough DNA for sequencing or for checking the success of a cloning step.
 
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lemonie3 years ago

Do you put dry-ice in Eppendorf tubes?

L

(I used to)
cbm104 (author)  lemonie3 years ago
No only undergrads
cbm104 (author)  cbm1043 years ago
Erm, I meant only undergrads can get away with it, what with health & safety etc (not that i only put dry ice in undergrads).
lemonie cbm1043 years ago

I was a naughty post-grad then...?

L

(we had a few fires...)
kelseymh3 years ago
Woot, more science on Instructables!

Given the way I'bles is designed to be visual, it'd be nice if you added a couple of pictures to the first and third steps. For example, a picture of a commercial PCR machine, a diagram showing the stages and cycle of the reaction itself, or some such thing.

At the end, maybe a picture showing an actual result from your amplication (e.g., a gel scan, annotated to show the A. thaliana 256 bp stripe). That way, non-molecular biolgists will have a sense of what they might see if they try this at home.
cbm104 (author)  kelseymh3 years ago
Cool, thanks! You are right about the gel pic, I'll see what i can do.