Useful as a quick easy method for large amounts of good quality DNA from
fish blood. Can be scaled down but as fish red cells are nucleated be sure
to to get a sufficiently small amount of blood for a miniprep.
Fish genomic DNA preparation
Take 100-300µl of fish blood and add to 10ml of 50mM Tris/HCl, pH
8.5, 20mM EDTA, 200mM NaCl, 0.01% SDS, 20µg RNAaseA in a 50ml polypropylene
Universal. Gently mix (eg rotary or rolling on a shaking platform) for
15mins. Add 10ml of phenol, pH 8.0 and gently mix for 1 hour (or more)
Spin 3000g for 10min and remove supernatant with truncated blue tip or
10ml pippette to a fresh tube. The supernatant will be viscous, stringy
and cloudy and will be difficult to separate from the interphase crud.
Dont worry too much just do your best.
Add 5ml of 50mM Tris/HCl, pH 8.5, 20mM EDTA, 200mM NaCl, 0.01% SDS and
mix gently. The stringy cloudy solution should go clear. Extract with an
equal volume of chloroform for 1 hour as before. Spin again and collect
10ml of supernatant as before. Be more careful this time- avoid the interphase.
Add 2 volumes of ethanol and invert several times. Pick up the high molecular
weight DNA on a bent pippete tip.
Slide DNA of the tip into a 1.5ml microtube containing 1ml of 70% ethanol
and leave for for 5 min. Spin for 1min at full speed in microfuge, decant
supernatant, pulse spin down the excess liquid and remove with a pippete.
Leave to dry for 5min (ie lid open on the bench). Add 0.5 to 1ml of sterile
distilled water (or TE). Leave in the fridge overnight to rehydrate, gently
mix next day and store as required.
Determine the DNA concentration spectrophotometrically and store at -20°C
to 4°C. This DNA is usually high Mwt and good for all purposes.
Notes - Do not use more than 300µl of blood in this protocol.
Scale up if required. 300µl of fish blood should yield at least 0.5mg
of DNA. Contamination can be a problem in Southern blot experiments where
large amounts of plasmids are being routinely used as very small amounts
carried over from, for example, a contaminated tip can overwhelm a Southern
blot. Therefore all reagents should be made up from scratch, autoclaved
and marked GENOMIC DNA ONLY and only fresh tips should be used. The same
points should be noted when restriction digesting and running the DNA ie
ideally seperate enzymes and buffers should be kept for this purpose.