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2012 - and 20 years of PCR
Happy 2012 to everyone. Of course I relate most things to PCR stuff (well, this is a PCR blog) and this year marks 20 years since I first did a reaction. Which means it's 20 years since my first blank gel and 20 years since I amplified strange bands, wrong-sized bands - before hitting the conditions and getting it right.
Or at least for the positive control. . .
Things I remember?
- While developing a new method, being given a tube of Taq, buffer and MgCl2 and following a paper (it used the same single primer)
- PCRs then were in 0.5ml tubes and about 100µl volumes
- Since I was only running 10µl on a gel, I figured I didn't need the other 80µl immediately chopped all volumes 5-fold (including the enzyme) to 20µl reactions
- 2 drops of oil as the overlay (yep, no heated lid). Plus greasing the tubes with the same oil on tissue paper before seating over the machine with the car headlamp as the heating element.
- Another student speeding up the tube greasing by dropping oil straight into the wells. Since the wells weren't solid, much smoke resulting as the oil heated on the lamp!
- Back then Taq was about $1.20 a unit. Now we're under 20 cents a unit (check out FIREPol)
- No hotstart enzymes. Hotstart - when I used it - was adding the MgCl2 to the reaction tube during a pause in the first annealing step.
- My positive control was just under 3kb. That was considered a long amplicon and I was asked from time to time how I got my 'big PCR' to work by others (ironically, low enzyme amounts would later be shown to be useful for longer PCRs). But then we were amazed to read of enzyme blends going out to 35kb.
I consider myself lucky to learn about PCR then and learn some of its fun attributes. Thanks Iain.
(I would later take some small perverse delight in sending papers to my former supervisor that made the method I developed obsolete)
You still remember your own first PCR reactions? Please share your memories. . .