[DIYbio] Re: First full DNA extraction, PCR, and gel

I looked at the gel picture and it seems there is a linear shift in bands from the left side of the gel to the right. Could be why the bands appear larger in size. Also, because of your short extensions times and the inefficiency of heat transfer from the "Air PCR" your extensions might not be completed. Usually when people run PCR they put a final extension step of a few minutes to allow any unfinished reactions to amplify, this might help.

There are usually lots of primer dimers, weird bands at the bottom of gels usually no need to worry much about optimizing to rid yourself of them. Instead invest in a PCR clean-up kit.$69 for 50 reactions, save you alot in the long run and the columns can be reused by soaking them in an acid and washing them a few times.
http://www.zymoresearch.com/dna-purification/dna-clean-up/pcr-dna-clean-up-concentration/dna-clean-concentrator-5

On Tuesday, December 25, 2012 1:36:08 PM UTC-6, Dakota wrote:

Hey all, I wanted to share the results of a gel I had run with a friend in the hopes of getting some insights into two questions we had, which can hopefully make the final writeup of the piece that much more complete, and hopefully of use to others. We managed to do the entire DNA exaction, PCR, and gel using (almost) all our own equipment and reagents we've been slowly gathering and so it was a proud moment! (Minus the water bath and micro-pipettes which weren't ours).   We basically wanted to make sure the PCR machine and microcentrifuge we got off ebay worked, as well as hone some basic wetlab skills.  

http://gyazo.com/b73e8c7eb29e45b87196fee9fbd86afa

That was the gel, 1% agarose stained with GelGreen under UV light.  The ladder used was a 1kb NEB ladder http://www.neb.com/nebecomm/products/productn3232.asp

A button mushroom (Agaricus Bisporous) was purchased from the store for 16 cents, and a plant genomic DNA prep kit from Epoch was used on a small sample from both the cap and the stem of the mushroom (freshly cut in half).

We used two different pairs of primers, ITS1 / ITS4 and NLB4 / NSI1

We expected products of around ~700 for ITS and ~900-1kb for NLB/NSI  but these can vary for different mushroom varieties (ie. Basiodiomycetes vs ascomycetes etc) 

We tested our PCR machine, an Idaho Technologies RapidCycler  vs a Peltier effect machine, neither of which had "heated lid" capabilities and so mineral oil was used.  

The Idaho tech one is basically a giant halogen lightbulb that uses a tornado effect to circulate hot air, then opens a hole in the lid to allow hot air to escape, and leads to really fast ramp/cooling times.  (After seeing it in action, I don't know why this technology isn't more popular than peltier).  If you use the glass capillary tubes, you can run programs to get products under 1kb in about 15 minutes total for 30 cycles...insane!!!  

Our program was

94C for 30s

54C for 45s

72C for 45 s

30 cycles (we wanted 35 but time didn't allow for it)

Even at 30 cycles and those program times, the Idaho Tech machine finished ~30 minutes before the Peltier machine, that was using a 25uL final rxn in 0.200 mL thin walled plastic PCR tubes with mineral oil.  I think with that machine (and maybe a peltier) you could get the total run time way down, as it took an hour and a half +. 

I also think the gel could have run a little longer for ladder band separation but again, we were running out of time at the uni lab we were using.   We ran the gel at 120V for maybe 25 or 30 mins.

QUESTIONS:  

So 3 things stood out in that gel.

1. Primer dimers (very faint in the set of primers on the right hand side, very noticeable with the ITS primers.)  

2. Our PCR machine seemed to make products that were slightly "higher" in the gel than the peltier machine...no idea why

3. Very intense bands on the right hand side in the NSI/NLB Peltier product lanes.

Primer dimers I'm not that worried about at the moment, though we do plan on sending this out for sequencing on Friday, and assume the $2 "cleanup" fee assessed will remove those.

I had thought that maybe running in the center of the gel had caused products in the center to run a little higher, but after looking at the ladders, they seem to be pretty even, but our PCR machine products are still slightly higher (so longer?).

Also, I was wondering if the really intense band in the right hand lanes was just a really good rxn, or if incomplete elongation had cause products which were a little shorter than the desired product, leading to a fattening of the band on the underside.

The primers used are below.

NSI1 (forward)  GAT TGA ATG GCT TAG TGA GG 

NLB4 (reverse)  GGA TTC TCA CCC TCT ATG AC 

ITS1 (forward)  TCC GTA GGT GAA CCT GCG G 

ITS4 (reverse)  TCC TCC GCT TAT TGA TAT GC

Thanks for any feedback!

-Dakota

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