Various DNA isolation techniques.

• DNA isolation from yolk sac
• Extraction of DNA from parafin sections

DNA isolation from yolk sac (crude for PCR).

From:Dev Genes Evol (1997) 206:377-388

Lysis buffer :

KCl	50mM
Tris-HCl pH 8.3	10mM
MgCl2	2.5mM
Tween 20	0.5%
Proteinase K	100ug/ml

Digest for 1 hour at 55 oC

Use appropriate amt. of buffer for the age of embryo.

Incubation can be longer and for larger yolk sacs I usually keep them moving by putting them in a rotary incubator.

Incubate for 10 min at 95 oC.

Use 5ul lysate in PCR reaction.

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Extraction of DNA from parafin sections (Newsgroups).

I extract DNA from PWEM as follows

Selct area of interest (i stain the slides using standard Haemotoxylin/eosin
protocol...Staining does not affect PCR)

Put the tissue into a tube with 100-500 microlitres of a proteinase K buffer
containing 0.1% tween (tween doesnt inhibit Taq you could use SDS if you are not
using PCR) and 0.5 micrograms/microlitre of Proteinase K.

Incubate the tube at 55~C for 4 hours.

Heat reaction to 85~C for 8 minutes and snap cool on ice

Use 1- 5 microlitres per PCR reaction

Good Luck

Stuart Pritchard
[email protected]