Sheffield Molecular Genetics Facility Protocols
http://www.shef.ac.uk/misc/groups/molecol/smgf.html
http://www.shef.ac.uk/mgf-s/protocols.html
If you can suggest improvements to this protocol please email Deborah Dawson
(D.A.Dawson@Sheffield.ac.uk)
Guidelines for taking samples from birds and mammals for DNA extraction
Birds
Bird blood is nucleated and contains a lot of DNA when compared to un-nucleated mammal
blood. This allows us to obtain sufficiently high quality and quantity bird DNA from blood
samples using relatively simple extraction techniques.
We have experience of extracting DNA from blood that has been stored in several different
buffers and we strongly recommend using ethanol and no other method for storing blood. We
definitely do not recommend storing blood in Queens lysis buffer or any other method which
uses an aqueous buffer and then requires freezing or storage at 4 degrees. If Analytical Reagent
grade ethanol is not available or limited due to tax reasons (as on some tropical islands) then we
recommend using DMSO (Dimethyl Sulfoxide) however DMSO is toxic and strict precautions
are required when dealing with this buffer.
Approximately 50 microlitres (one drop) of blood should be added directly to 1ml (1000
microlitres) of absolute ethanol (must be Analytical Reagent grade) in an eppendorf. The screw
top should be securely sealed and then the tube shaken well to mix the blood and ethanol.
Eppendorfs are then stored at room temperature.
The ratio of blood to alcohol should not exceed 1 in 10 - this also applies when collecting tissue.
50 microlitres of blood will produce more DNA than could ever be used in a normal genotyping
study. Therefore collecting more than 50 microlitres is not necessary. If excess blood is collected
and the ratio of blood to ethanol is reduced the blood will become degraded and be of little use
(less DNA will be obtained following extraction and this DNA will be degraded causing
problems for genotyping). If samples have been overfilled they should be stored in a spark-proof
freezer to prevent degradation. It is very important the freezer is spark-proofed because
otherwise the alcohol in the sample could cause an explosion.
The eppendorfs used should be the type which have rubber sealed lids. We use those supplied by
Starsedt. The eppendorfs and their lids should be clean and dry and need to have been autoclaved
to ensure they are sterile. After adding ethanol and the blood sample ensure the lids are secured
as tight as possible otherwise the ethanol will evaporate. Use autoclaved time tape (or autoclave
tape) to label the eppendorfs and ensure you use a permanent marker to label the tube. If
transporting eppendorfs containing ethanol and blood the lids should be double wrapped with
parafilm (nescofilm) to prevent leakage.
Notes
Heparin will prevent or inhibit PCRs so it is important that when taking blood samples heparin
coated capillary tubes and eppendorfs are NOT used.
It is important not to contaminate the blood sample with blood from other birds or with your own
human DNA so you should not blow blood out of capillary tubes. Blood can be released into the
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Sheffield Molecular Genetics Facility Protocols
eppendorf by taking your finger off the end of the capillary tube and letting it flow out gently
with gravity. This works 90% of the time, if the blood doesn't flow out, you can use a little puffer
that fits over the end of the capillary tube and you squeeze to blow the blood out the other end.
Suppliers
Eppendorf tubes for small amounts of blood:
1.5ml micro tubes screw-cap enclosed, cat # 72.692, pack of 5000, £77.80 (2008 price), Sarstedt
Ltd.
Absolute ethanol (100%), Analytical Reagent grade, duty free, 2.5litre, cat# E/0650DF/17,
Fisher Scientific UK Ltd.
Bird corpses
Blood is also the best sample to collect for DNA extraction from dead birds (if the bird is freshly
deceased or has been frozen soon after death). Bird blood is nucleated so contains a lot of DNA
whereas little DNA is obtained from muscle tissue, foot pads or feathers. If no blood is available
brain or liver are the best tissues to collect from bird corpses - but blood is always best and is
easiest to extract from - assuming the bird corpse is relatively fresh - or has been frozen soon
after death. The tissue should be diced and stored in ethanol as above (ie Analytical Reagent
grade ethanol, not more than 1 to 10 ratio of tissue to ethanol and stored in rubber sealed tubes).
Feathers
Feathers with large rachises or vanes (contour or flight feathers) will yield the highest quantities
of DNA, while freshly plucked feathers provide the highest quality DNA. Moulted feathers
provide DNA with varying degrees of quality and quantity. Those feathers with no visible signs
of degradation, a transparent calamus and intact barbs on the vane will yield the best results.
DNA can be extracted from two parts of the feather: 1) 5-10mm of the basal tip of the calamus 2)
a small, usually visible blood clot found where the vane meets the calamus (superior umbilicus).
Higher quantity and quality DNA can usually be obtained from the latter. Where possible,
several feathers (2-3) should be taken from each individual. Feathers from different nests or birds
should be stored in separate bags/envelopes. Feathers do not need to be frozen and can be stored
dry at room temperature.
Mammals (blood, tissue and faeces samples)
Mammal blood is not nucleated so a larger volume is required. Alternatively tissue samples
should be taken instead. Liver or brain is normally used from animal corpses.
Tubes for larger amounts of mammal blood or tissue
Tubes - 13ml round bottomed tubes+screw caps + O ring, Case of 500, closure enclosed, £90.00,
cat #60.540.016 PP, Sarstedt Ltd.
For storing dry mammal faeces (ie without ethanol)
silica gel self indicating (orange1-3mm), 1kg, £28.22, 1.01969.1000, BDH Ltd.
Ethanol will cause some plastics to crack and shatter. The type to go for is polypropylene- not
polystyrene. A rubber sealed lid is again necessary to stop ethanol/sample leakage.
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