Streamline Your Cloning |
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Emily Crow |
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I always keep an ear open for helpful tips in the lab – those little tricks that can make your experiments faster, easier and better. Here are a few tricks I’ve picked up for trimming down the time it takes to do your cloning:
Restriction digests
Many digests are complete within 10 minutes of digestion at room temperature – no need to be a slave to the “one hour at 37°C” rule. Check in the back of the NEB catalog for their list of high efficiency enzymes that digest quickly, or test your enzyme by yourself to minimize digestion times.
PCR amplification
There are a few ways to cur down on the time it takes to run your PCR steps…
Ligation
Ligations can work surprisingly quickly: T4 DNA ligase can complete a reaction in just 10 minutes at room temperature. Check the product literature for your enzyme to see how short of a ligation you can get away with. Keep in mind that difficult ligations will generally be more successful if you allow the ligation to proceed longer.
Transformation
Just about every step of E. coli heatshock transformations can be shortened…
All these changes will affect the efficiency of the transformation, so use them wisely. Difficult or low-frequency ligations need some coddling, so it is probably best to use the full-length steps in these cases, although you might get away with some shortcuts here if you use very highly competent cells.
Agarose gels
What are your favorite tips for speeding up your cloning?
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Get Your Clone 90% Of The Time with Ligation Independent Cloning
Ligation Independent Cloning Protocol
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James Lloyd
Since joining my current lab, many of us make up about 400ml of 1% agerose and store at 65oC in incubator and leave until needed. just pour, set and run. not as fast as the home made pre-cast gels above but they do store for over a month if you dont keep them out of the 65oC incubator for too long and doesnt take up as much room.
Pete
Cloning or none, I’ve never had a satisfactory answer as to why you would need the final elongation step in a PCR. As far as I can see, if previous elongation steps create truncated fragments, then subsequent steps would have no _new_ template to amplify. If you run 35 cycles + the final elongation, this would still result in only a 36-fold amplification, rather the full 2^35 = 34 billion-fold.
Even if elongation of some molecules work and some don’t the principle would still apply. Truncated molecules hardly affect future cycles. Surely it’d be more worthwhile to stick an extra 17 seconds onto each of your 35 cycles rather than 10 minutes at the end.
…and as for the initial extra-long denaturing step…!
Jode
Pete,
The final elongation step was described to me as a polishing step to improve the clone-ability of the fragment. The idea is that the polymerase gets a little flaky at the end of the DNA molecule. While leaving off a handful of bases still allows that strand to anneal to a new primer and extend in the next step, it could screw up your restriction digest or not leave blunt ends when you were counting on them for cloning. That final elongation gives the polymerases time to fill in these missing bases. It’s possible that this step is a legacy and isn’t needed for the current crop of polymerases, but it doesn’t seem worth it to properly test that idea.
Matt
Using sodium borate gels, referenced here here, we routinely run agarose gels at 350V in about 7 minutes.
Also your choice of enzyme can affect how your run time as well. We used to use Vent polymerase from NEB for PCR reactions and Taq for colony PCR. We have recently switched everything over to Phusion (sold by NEB, made by Finnzymes) and its processivity is 4X as fast as Vent or Taq and its error rate is also extremely low. Phusion can polymerize 1kb in 15 seconds versus 60 seconds for Taq or Vent.
epibio
One factor that’s often overlooked when cloning DNA inserts that have been gel-purified is the effect of UV damage on cloning efficiency. We’ve found that even a 30-second exposure of DNA to UV light on a transilluminator decreases the number of transformants obtained using that DNA by more than two orders of magnitude.
A good alternative is to use a Dark Reader (Clare Chemical) instead of a UV transilluminator. You can also run a duplicate gel lane, cut it out, stain and visualize the band with UV, then use it as a marker to locate the band of interest from the rest of the gel that’s not exposed to UV.
Peter
Good article, Emily! I’ve been streamlining my own cloning tasks for a while… Here is my addition to the list. Use a quick silica bead gel extraction kit (like the one from Fermentas) for DNA fragment isolation or cleanup. It takes only about 10-15 minutes hands-on time to clean up and concentrate DNA fragments from a piece of agarose.
Caroline
I rarely incubate my cells for more than 5 min with the DNA. I don’t know where this 30 min post-thaw comes from. Does anyone know why you would leave the cells on ice that long before heat shocking? Also, I can second the blue light vs. UV. My cloning efficiency is phenomenal now that I have switched to a dark reader. I would often have to screen 10 clones, now every colony is positive. At least that’s been the trend for the past 10 ligations.
s_laub
When loading a gel, no need to change tips unless you plan on cloning the fragment.
If a ligation is really pissing you off, try PCRing from the ligation reaction the fragment you’re expecting. If you don’t see it, you don’t have it and you can quit the screening process and re-examine what’s going into the ligation.
I have what I believe to be the best miniprep procedure. It involves scraping cells from a plate into the “easyprep” buffer in an ependorff, shaking for 5 min, boiling for 1 min, ice for 1 min, spinning fs/RT/20 min and using the supernatant for ANYTHING. I routinely use this to screen as many as 54 colonies. Then I run this on a gel and then digest each “size class” of the plasmid. I can usually find what I’m looking for in a single day (after the o/n growth time of course) and this often takes me about 5 hours including gel run time, digestion, etc. If anyone’s interested I’ll post the recipe. The DNA keeps for quite some time at RT (I’ve gone back into the garbage after a weekend to dig up some old stuff and it’s still good – at 4*C probably a month – plenty of time for a good old fashion screening). I’ve used it for PCR and apparently it can be used to transform directly and even for sequencing.
Casper
I just learned a very simple trick. It always takes a long time to cool down your agarose gel after microwaving to add the Ethidium bromide and poor the gel.
Just weigh you agarose and add half of the required volume of buffer. Microwave to disolve the agarose and than add the other half of your buffer. Tadaaaa, you gel is at the right temperature to add ethidium bromide and immediately poring.
Emily
Fantastic suggestions – I’m looking forward to trying them out on my next cloning project!
I’ve also recently become indoctrinated into the “in-gel ligation” school of cloning, which saves me 15 min of gel purification. An older technique, I think, but new to me!
Roman
for Casper:
You don’t even need to let it cool down!
As you described, I learned that EtBr is not heat stable, but nowadays I’m just adding 2-3µl EtBr to 80ml of TAE/Agarose just when it comes from the microwave and pour it! That’s no problem at all!
sashko
s_laub, I’d like to know more about your miniprep procedure.
What’s the composition of your “easyprep” buffer? How big do the colonies need to be before you can screen them? It would be amazing to save a day of waiting for minipreps to grow…
John
The final extension step in PCR? Indeed, published a while ago that TA cloning eff increased with a (from memory) 30 minute (!) final extension. I always believed it was to increase band sharpness on gel so that any shorter products were making it fuzzier. Probably all bollocks
sebastian
s_laub, I would also be very interested in reading that recipe for your ‘easyprep’ buffer
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Alejandro Montenegro-Montero
For simpler cloning (with less handling time and high efficiency), you can always try in vivo recombinational cloning (check my original blog post in the matter here:http://amontenegro.blogspot.com/2009/12/alternative-cloning-strategy-yeast.html, and my article here: http://www.lab-times.org/labtimes/issues/lt2010/lt03/lt_2010_03_66_67.pdf)
Shana Garrett
Matt, What type of box are you running gels in at 350 V? Our biorad boxes crack at 100V…We have one OOOOOOLLLLDDDD Home shop fabricated box that will not splinter at high voltage.
Matt
Shana,
All we used to have, before we got in to a lot of cloning, was a very small biorad box. We then borrow some Owl electrophoresis boxes from a sister lab and upon finding out how much they were my jaw dropped. The price for some sheets of acrylic, two banana plugs and some wire is amazing (must be special science plastic).
In short, I made two of my own electrophoresis boxes for less than $30 each. If anyone in your lab has a miter saw, a weekend, and a desire to try new things then my suggestion would be to fabricate your own box.