First full DNA extraction, PCR, and gel

[Dakota]

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

[Matt Lawes]

Hi Dakota,

Thanks for posting the gel. Generally nice data! In my semi-pro opinion, the NLB products from both cyclers are the same size, but the higher yield from peltier (probably due to more cycles) creates the effect of slightly faster migration. The DNA product is bunching up and there is so much there it's changing the ionic environment in the gel relative to the buffering ions ..... so it runs a bit faster during electrophoresis.

>matt

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[Dakota]

Thanks for the response Matt, makes sense.  Now that you mention it, I had forgot that when I programmed the old peltier cycler, I did repeat step 1 30 times, when I think I should have done 29 times, to make it 30 in total.  So...I think the peltier machine actually did 31 cycles, not 30, so it could be the reason for the brighter bands in the peltier lanes.

[Matt Lawes]

I think you're looking at more than a two fold difference in yield. Perhaps 10 - 50 fold more. Suggests that the Peltier is more efficient ( meaning better extension, doubling of more cycles). May be the trade off from the rapid cycling with the Idaho machine. The average Taq polymerase will extend 1 kb target in about a minute .... so maybe the short 45 second extension at 70 - 72 is penalizing yield in the rapidcycler? Less of an issue with the shorter first target & primers.

Room to play a little to optimize yields with the rapidcycler.

>matt

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From: Dakota <dko...@gmail.com>
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[Dakota]

Yeah some optimization is definitely in order, but it should be enough DNA to sequence at least.  The Idaho machine is designed to use glass capillary tubes, but ours came with a tray which I believe is for thin walled normal PCR tubes.  The capillary tubes specific for this machine are discontinued, and the company that makes them, Bio-Fire or something, said they may or may not have them in stock.  Though I can probably get my hands on some, and loading them won't be a problem with capillary action, the after process seems to be a bit of a pain.

You have to flame seal them like an ampule, which removes the need for mineral oil, but is an added step, and then to remove the sample from the tubes you need some special needle aspirator or tiny pipette thing that costs $100 + who knows how much for tips.

When I was reading about the rapid cycler system, they said that you could do 1kb or less product amplifications with something like a 0 or 1 second denature and anneal time, and a 10-15 second extension time, if you use the glass capillaries.  

[Cathal Garvey]

Wow, I'd never even considered that as a factor in gel migration! Cool! :)

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[John Griessen]

On 12/30/2012 04:58 PM, Cathal Garvey wrote:
> After seeing it in action, I don't know why this technology isn't more popular than peltier

It will be when I get my "air pcr", (named by Bryan B.), machine productized.

[Dakota]

Yeah I was pretty impressed with it, it did a great job for being $50 off ebay.  I havn't really looked at heating ramp times compared to peltier, but when the lid in the top of the machine opens, it cools very fast.  The only downsides I can think of quickly is the need for mineral oil as well as not being able to do a 4 degree C final hold.

But, if you can give up those two things, it saves a lot of time.  After we get the sample sequenced, going to re-do some amplifications and drop extension and annealing times by  5s or 10s a run and still see if PCR works. 

I re-did the write-up and posted it, http://basementbiotech.org/  but it's basically the same, with just an extra picture of two.

I'll be one of the first in line if AirPCR comes out!

[Patrik D'haeseleer]

Sounds like the main bottleneck with thethe Rapidcycler is the heat transfer from and to the sample - hence the use of glass capillaries and mineral oil. If you're using plastic PCR tubes, you should count on a much longer ramp time. That's probably why the Peltier was more efficient.

Patrik

[Dakota]

Yup heat transfer is the key, but you can avoid mineral oil with glass capillaries since you flame seal them into a closed environment.  Getting the samples out of the capillaries requires some weird aspirator needle thing though, supposedly.  

http://www.biotechniques.com/multimedia/archive/00001/BTN_A_000112722_O_1702a.pdf

It does go to show though, that with the glass capillaries you can do a full 30-35 cycle runs in 10-30 minutes, but they were only amplifying a 250bp target.  

http://gyazo.com/136119c336f07381fc1183c06753e259

That's the chart that shows plastic vs glass, and below you can see how fast the program times are, and as you say, much higher lag with plastic in red.  Pretty nifty stuff.

[John Griessen]

On 12/31/2012 12:49 PM, Dakota wrote:
> Yup heat transfer is the key, but you can avoid mineral oil with glass capillaries since you flame seal them into a closed
> environment. Getting the samples out of the capillaries requires some weird aspirator needle thing though, supposedly.

Glass is a fine material. You know how incandescent lamp bulbs are made in various controlled shapes...so,
is there a source of glass vials 6-8mm diameter that could be capped with plastic snap on lids?

Hmmm.... yes, here are 200/$36, or about 0.22 USD each:
http://www.sigmaaldrich.com/catalog/product/aldrich/z291994?lang=en&region=US 8 × 40mm, 1mL vol. Constructed of clear
borosilicate glass. Vials come with LDPE snap-plug caps.

These would be good to design a system around. I'd prefer a little shorter than 4cm though.

Here are shorter ones. http://www.biotechsolutions.org/vials.htm

[Dakota]

Found the old handout I had seen months ago!

http://dna.utah.edu/References/PDF_Public/RapidCyclist_V3_1995.pdf

In that one you can see the capillaries look like melting point ones,
and involve drawing it up with capillary action then flame sealing and
removing with that syringe thing I was talking about.

Those glass ones you posted look pretty nice, would probably improve
the efficiency with the setup I have now by ditching the thin walled
regular plastic PCR tubes.

http://www.biotechniques.com/news/The-ups-and-downs-of-convection-PCR/biotechniques-307800.html

That link applies to the ones you linked, they look identical.

[John Griessen]

> http://www.biotechniques.com/news/The-ups-and-downs-of-convection-PCR/biotechniques-307800.html


They say, "As the sample entered the cool zone at the top of the tube, it underwent annealing and extension. Then, the DNA dropped
to the bottom of the tube and was heated again. "

I think they are saying DNA expands to the point of being suspended when hot,
and sinks when cooled, or are they just saying some toroidal flow patterns happen
in the size tube they used?

[Nathan McCorkle]

pretty much what lava-amp was going for, there was a lot of talk about this sort of thing a few years ago but it never picked up mainly because they were all focused on using circular capillaries that were either expensive/hard-to-get or weird/hard to work with (someone mentioned in a recent thread regarding annoying capillaries for an air thermocycler)

http://www.lava-amp.com/

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-Nathan

[Nathan McCorkle]

I also have a air thermal cycler, I haven't tried it out since I bought it from an electronics recycler... it's made by Hybaid (some british company that seems to have been bought up)... I paid $40 without the thermocouple and here they're offering it with a thermocouple for $849!!! I think I can use any K type thermocouple, but I haven't found a cheap supplier for them... thus the reason the unit is sitting. (well and that I haven't been doing PCR). I recently bought a chinese electronic multi-meter that came with a thermocouple, maybe I'll play with that this weekend.

http://www.labextreme.com/detail.cfm?autonumber=54919

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-Nathan

[Josiah Zayner]

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

[Nathan McCorkle]

I would think having lots of primer dimers would mean you could use less primers earlier in the reaction, unless your template was really low concentration (higher concentration of primers in the beginning of the reaction would increase the chances for primer and template interaction) 

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[Dakota]

Yeah next time around probably going to aim for 60s extensions, but
drop the anneal and denaturation to ~10-15s and try to use some glass
tubes as well as plastic. Also never did an initial long denaturation
step which I see sometimes used, or a final extension like you said.
I havn't messed with the interface and programming on the PCR machine
much but plan on reading over the old manual, was in a rush that night
to get everything done in one go. Just wanted to set a baseline of,
"ok this works at least" and then go from there as for fine tuning.

Just put them in the mail this morning at 10am for sequencing, forward
primer only, so we'll see how the data comes out.

As for primer dimers, could take a look at the concentrations but they
were the same for both primer pairs with 0.5 uL @ 10uM concenation.
Notice the ITS primers were the only one with dimers, the other pair
weren't as noticeable, if at all. So that might suggest it's
something besides concentration, but perhaps my friend messed up
calculation when he made them and they are at a higher concentration
then listed...or since I did the ITS primer set while he did the NS1
set, maybe I messed up somewhere along the line in how I setup the
reaction.

I have a PCR cleanup kit but, got 2 free sequences from GENEWIZ for
normal / custom order, so figured just go with custom and let them
clean it up and figure out concentration with their nanodrop or
whatever they use. This'll be the first time we'll ever get our own
data to use with BLAST, so hoping to learn more about the
bioinformatics side, I'm sure that's an entirely different beast.