Extracting Spider/Bacteria DNA Using Columns – Spider Silk Step 1

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The Great Courses Plus Doing biology work is a lot like cooking You’re always following a recipe
but there are countless potential tweaks and variations you can make
to change the final outcome With biology every technique
has half a dozen variations And the ingredients are made by
dozens of manufacturers each with their own custom blends
of compounds for the task at hand Think of biological reagents like
barbecue sauce A recipe may just call for barbecue sauce
but there are dozens of brands and flavors and your choice can play
a big role in how the final dish tastes So, most lab work requires some knowledge of which protocol variations and particular products work for your specific experiment Take electrophoresis for example Using the same basic recipe you can perform
all of these different blotting experiments on a huge assortment of molecules or with PCR there’s likely
dozens of different types based on the DNA source and
what you’re trying to do to it As well as a ton of other subtle factors What usually ends up happening
is you shop around look at all the different
products and protocols and see if you can find one that
says it does the thing you’re trying to do Then you order those supplies,
try it out, and see what happens. Maybe it works right off the bat but often you’ll end up having to
try a couple others before you find one that gives you good results Today we’re going to look at
one central recipe And some of the variations we’ve
been using over the past few months If you’ve been following along,
you’ll know that we’re working on engineering a strain of yeast
to produce spider silk One of the first steps in that process is
getting some DNA out of the spiders so we can isolate
the silk gene itself This has proven to be
a nightmare and to date, I’ve used almost a dozen spiders
in various attempts So today we’re going to explore and compare
successful DNA extraction procedures for two different DNA sources Some bacteria And the spiders Before we go further let’s go over the basic
cake recipe before we talk about flavours we’ll be using a column based approach which tends to give better results than the shot glass
and alcohol method you may have seen before It’s not to say that
that can’t work but this method is more gentle and keeps the DNA intact better as well as being
standard lab procedure there are also other variations,
like bead-based methods and we’ll look at some of those
in future videos All of these extractions work
on a similar series of steps First, we get the thing we want to
extract DNA from and make it into a sort of paste
and add a salty, soapy solution to it this pops open all the cells so that the
DNA is floating around in the salty water we then remove as much of the debris as we can be it bits of broken cells,
or a leg, or whatever else This is done using a centrifuge, and all the
heavy debris will sink to the bottom We then take our clean solution
and load it into this special tube It’s basically the world’s
smallest coffee filter and the filter, in this case, is either
made of silica or a special resin we load the tube into a larger tube which will act as our collection cup and then spin it in the centrifuge to
pull the liquid through the filter DNA is negatively charged and in
the salty conditions of the solution the silica or resin becomes positively charged so the two stick together when we spin down the column the rest of the solution containing everything
else just passes right through we then do a series of washes
to clean everything and then we’re ready to collect the DNA we transfer the filter to a clean tube and then add a small amount
of either sterile, distilled water or various other solutions so long as they’re not salty without the salt, the silica’s charges
get hidden and the DNA falls off this time when we spin everything down we end up with a tiny amount of
concentrated DNA solution most of the time when you do
this you buy all the tubes as a kit that come with all the different
solutions you’ll need the manufacturers tune the solutions in
each kit to work for their particular application so if you try to mix and match,
your results can be pretty poor right now I have two different kits and I even tried mixing up my own solutions
though that didn’t work very well Let’s start with the bacterial kit This one is made to isolate plasmids which are small circles of DNA that we
add to bacteria to make them do new things sort of like putting a CD into a computer in this case I grew some E. coli that have a
plasmid in them that makes them bioluminesce but this should work regardless
of what the exact plasmid is the plasmid uses tetracycline
as the selection agent so I prepared 1 milliliter of LB broth with 10 micrograms per milliliter of tetracycline and innoculated it with some bacteria
and let it grow overnight on the day of the extraction I first spun down this liquid
to collect all the bacteria into a little pellet at the bottom the LB was removed and then it was on to the
various buffers and solutions in the kit The kit just labels things like
“PD1” “PD2” and “W1” which doesn’t really tell you what’s going on but I was able to find some more info online PD1 contains an enzyme called RNAse which, as the name implies,
destroys RNA this is to make sure you only isolate DNA PD2 is what’s called a Lysis buffer basically, it’s very salty, and contains a
special soap or surfactant in this case SDS as well as an enzyme, called Lysozyme which eats bacterial cell walls the salt makes the bactera shrink,
the enzyme eats away at the structual integrity of the bacteria and since cells are basically living bubbles of oil the soap makes them all pop open and whatever was contained inside is released into the solution you add both of these to your bacterial pellet and then re-suspend the bacteria
and let it sit for a few minutes so it has some time to work Once it’s done we add PD3 Which seems to crash out all the
proteins and debris from the solution making it go cloudy We spin this down, which collects all the floating
junk at the bottom, leaving our DNA in solution then we carefully pipette
off the upper liquid called the supernatant and run it through the filter using the centrifuge After it’s been run through the column
we wash the filter with wash buffer which contains a large amount of alcohol
to help remove any stubborn organics we then spin the filter while
it’s empty for a few minutes to dry it so all the ethanol is gone
before we elute the DNA For the elution, we add 50 microliters of
sterile water or elution buffer since the kit comes with that Rather than spinning it down immediately, we wait five to 10 minutes to give
the DNA time to release from the filter I’ve left columns for half an hour or more
if I really want the DNA to come off Then we spin it down to collect our sample You can actually run a second
batch of elution buffer through or collect the first batch and run
it through again to try and squeeze out
every last bit of DNA But that’s it, and the extraction is complete It’s pretty slow because of all the
waiting for things to spin but it can be done in less than an hour
once you get a rhythm going As I mentioned earlier, there are some
other variants of this same basic protocol and some forego the columns and instead you magnetic beads coated
in either silica or the resin Replacing some of the centrifuge steps with beads
can really speed this up That’s why I was working
on making some of my own I actually just got the supplies to make more So expect some tests of those soon Now, let’s compare to the spiders I actually tried running a spider through the bacteria kit and it failed as spectacularly as you might imagine The reason is because the solutions
are tuned to solve a different set of problems First, lysozyme, the enzyme in the bacteria kit
only affects components of bacterial cell walls So the much more robust spider cell walls
and clumpy proteins were unaffected On top of that, the spiders are full of
natural inhibitors and other enzymes that are still active So when you pop open any of the cells they can wreak havoc and destroy any
good DNA you manage to get out So I picked up a special insect/arthropod kit which addresses all of this It’s a 27 step procedure and some of these steps loop
or have long incubation times When I did this I started at 5pm
and didn’t finish until 11:30 not time efficient by any means
but if it works, it works First up, it only wanted 30 milligrams of tissue Which is about a third of a spider This may not seem like a lot, but
it’ll give plenty of DNA to work with I chose part of the dorsal side of the abdomen As it’s high enough enough to not touch the silk glands and make a mess but also contains lots of soft tissue this was suspended in CTL buffer and I used a P1000 tip to grind the tissue
and disperse it in the solution in this kit, this is the lysis buffer but instead of SDS as their soap
they use a cationic surfactant called CTAB Then I added an enzyme called Proteinase K As a protease, its job is to break down proteins And since spiders are chock full of them
it’s got its work cut out for it To speed things along the tube was put into a heat block set to 60 degrees Celsius This is the longest part of this process and can take anywhere from 30 minutes to 4 hours Before putting it in, the solution was murky and opaque but after two hours it had fully cleared which is the stopping point To really clean things up, the kit says to add a 24:1 mixture of chloroform and isoamyl alcohol Neither of which come in the kit So I made some chloroform and sourced the isoamyl What this will do is collect any non-polar debris as well as twist proteins inside out and move them out of the aqueous layer After giving everything a gentile mix The tube is spun down and
you end up with a layer of debris at the interface between
the water and chloroform I ended up making a stack of stuff so I could get this at eye level to make it easier Not great lab practice but I wanted good results The top aqueous layer is transferred to a fresh, sterile 1.5 milliliter tube for further processing Being careful not to disturb the debris To our now clean solution we add
some RNAse A and HBC buffer and incubate at 70 degrees Celsius for 10 minutes This will destroy any RNA while also deactivating any proteins that stuck around then we add ethanol before loading into the same sort of filter and tube setup as before However in this case the kit says their
filters are resin-based not silica based which I thought was interesting After spinning that down, it’s on to a series
of washes with the provided wash buffer And finally after all that we can dry the column by spinning it empty for a few minutes Transfer it to a final collection
tube and elute our DNA Again, letting it rehydrate for a few minutes beforehand As you can see it’s still the same basic process
but because of the tough tissue sample extra steps and ingredients were
needed to get the best results Speaking of the results, there are two ways to analyze your sample once the extraction is done Run it on a gel, which we’ve
discussed in two previous videos Or test it with a special spectrometer Personally, I like using a gel better Not only is it vastly cheaper, since we dye the DNA with a stain that really only sticks to DNA It’s much harder to get a false positive
which actually happened during an earlier spider extract The spectrometer said we had hundreds of nanograms of DNA But when we ran it on a gel,
there was clearly no DNA present This is because the spectrometer can easily be set off by protein or other chemical contaminants left over from the extraction We’ve since run both samples on a gel and we finally got lots of DNA out of the bacteria and spiders We actually saw the bacterial
result in the gel dock video But there’s a problem with the spiders In theory we’re supposed to be isolating genomic DNA which is huge strands of DNA As we discussed in the gel electrophoresis video this should look like a nice
band near the top of the gel But the spider DNA looks like a smear low on the gel, which means its highly damaged and most of what we isolated is
shredded DNA fragments This actually matches up with an earlier result where we managed to get a whiff of spider DNA but at the time assumed it had just been damaged by the extraction protocol Now the running hypothesis is that the ethanol preserved the spiders and prevents them from rotting But it doesn’t stop the spiders native
enzymes from destroying the DNA And this kind of makes sense When I cut into the spiders they
were mush on the inside meaning everything was damaged So now I’m looking to source fresher spiders. I’ve already got a few leads, so
hopefully some of them will turn out As a last resort, we may switch to another spider species because they’re easier to get But for now I want to see if we
can get the widows to work At least now we know that this kit works so as soon as we have a fresher specimen we can definitely move forward but other than that, those are all the basics of DNA extraction using columns Depending on the style and exact kit the amount of time it takes can vary, but
it’s never really a difficult process It mostly involves planning in advance, getting the right tools, and following the recipe Before we wrap up this is actually a great moment to talk about the
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a moment to talk about Nerd Thunder Which is an opportunity for science and maker channels to do some cross-promotion This time the channel I think you should check out is:
NileRed I know some of you already watch his videos but he did a great video on extracting
DNA with the shot glass method Though he used a whole one liter beaker. So be sure to check that out if you want to see how you can extract DNA without using any fancy kits and using only stuff you have
lying around the house And that’s where I think I’ll end this video If you enjoyed be sure to subscribe and
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