Dearll,

Manyhanks for the excellent replies to my query on extracting DNA from
ethanol stored bird blood samples. They were extremely helpful and I
have copied them below to redistribute to the list as promised!

Allhe best

Mark

Markavinet
Researcher
CEESslo
mark.ravinet@ibv.uio.no

***
Iave had amazing results using the Phenol:Chloroform extraction method.
I have been doing this for over 10 years and always end up with so much
DNA I need to keep diluting it! I do an ethanol precipitation at the
final step and re suspend my pellet in water.

***
Iave always used the proteinase K/phenol:chroloform protocol, and is
quite good (better than any kit I have ever tried). You just need to
clean the blood from the alcohol with some TE before starting…

***
When have to deal with avian blood samples in ethanol, I always have two
phases clearly separated in my tubes.  Iemove the most ethanol I can,
then take blood cells (with fine tweezers for example) and put them in
the new tube I will use for extraction. I let this tube open for a few
minutes to evaporate ethanol (which could interfere with the extraction
steps).  Then use the Qiagen DNeasy Blood and Tissue standard protocol
(for nucleated blood cells

***
Iave used two protocols for this. Usually you get the highest amount with
a phenol-chloroform protocol. The problem I faced there was that I had
difficulties getting the DNA into solution again. You'll usually get
this with high molecular weight DNA. However, this was not a problem
for Illumina sequencing for the few birds for which we used this
DNA. For most samples I then used a modified protocol of the DNeasy
Blood and Tissue protocol. What proved important with this was to do
the proteinase digest in double the volume and to ensure the blood is
properly digested. For this, I usually put one volume of pK at 56°C an
hour or two before leaving for the night, and then added another volume
just before leaving and leaving the digestion run at 37°C overnight. If
there was still some coagulate left in the morning, I added yet another
volume of pK to 56°C until it was digested.

***
Ineneral, for high yield AND high molecular weight DNA, I usually go for
phenol-chloroform extraction, as it is efficient and can render really
good quality (if you’re careful).  Therotocol is optimized for blood
collected in SET buffer, but I’ve “modified” it for samples stored
in EtOH, i.e. picking out blood, quickly letting the EtOH evaporate,
and putting it in SET to proceed with the protocol. It’s important to
only letting the EtOH evaporate rather briefly, or the blood will turn
rock solid if it is left to dry for long. Now we’re on details, but
some blood samples form chunks similar to coarse sawdust: these samples
are great and simple to handle, pieces can easily be picked out with a
toothpick or similar. Some blood samples instead behave as more or less
dissolved, and these are a bit trickier. I usually centrifuge them down
properly, and use a mini shovel (I really have no idea about what the tool
is called, but something spoonish that is small enough to reach the bottom
of a tube) to scoop up blood along the wall of the tube. In either case,
just let the blood evaporate in the air, or gently press a tissue against
the sample to suck up ethanol (especially for the latter type of sample).

Now don’t remember the lysis step in the B&T kit, but
if you want to continue with that just put the blood in the
whatever-buffer-is-used-for-lysis and continue. This is at least what
I did some five years ago for some 200 samples used for RADseq, with
reasonably good results.

Therere also further optimizations that can be made for obtaining
ridiculously long DNA molecules, such as picking out the precipitated
DNA cloud from the ice-cold EtOH using a glass staff etc. Let me know if
you’re interested in a description of this and the subsequent washing.

(And,s you will also know, if you are going to RNase A treat your samples,
do it during the lysis step to avoid the risk of DNA degradation by
the enzyme [or make sure to boil it before use, as DNase most often
co-purifies with the RNase].)

***
Wetore most of our blood samples in ethanol.  It doesn't matter what
extraction method you use: chelex, kit, phenol=chloroform.  The main thing
is you need all the ethanol to evaporate before you start the extraction.
We typically take 10 ul of blood ethanol mix and put it in a 1.5 ml tube.
Leaving the lid on the tube open, put the rack in an incubator (37-55C)
for 15-30 min or leave it on the bench.  The ethanol will take longer
to evaporate at room temp, but both work.

***
Iope everything is going well with you. I have seen your
post in evoldir. Is it because you have experienced problems
with the DNA extraction? It is usually very straightforward
as well. I usually pick up a piece of blood with a wood
dental stick (but it should be flat). See for instance this
picture. http://www.tepeusa.com/products/wooden-dental-sticks Inpain,
you can buy them in the supermarkets and they are really cheap. Then,
I usually take around 3 mm of blood with the stick (I cut the tip first)
and I leave it on a paper lab for drying at room temperature. After
10 minutes (more or less) your sample will be ready to start with the
Qiagen protocol.

mark.ravinet@ibv.uio.no