Biology Reference
In-Depth Information
DNA bound
in membrane
Transfer
solution to
spin basket
Remove
swab
Centrifuge
(a)
(b)
(c)
(d)
A wash buffer is added to the spin basket and centrifuged:
this wash process is repeated twice, using different wash buffers
DNA bound
in membrane
Wash
buffer
Elution buffer
Centrifuge
Centrifuge
Centrifuge
DNA in
solution
(h)
(g)
(f)
(e)
Figure 4.2
DNA extraction from buccal cells using a salting-out method based on the QIAamp
Blood Mini Kit. (a) Cellular material is added to a lysis buffer and proteinase K and incubated
at 56
◦
C for at least 15 minutes. (b) Ethanol is added to the solution before it is transferred in
order to provide the optimum DNA binding conditions. (c) The lysis solution is then transferred
to a spin basket that has a membrane that will bind the DNA in the presence of the chaotropic
salt. (d) The spin basket is centrifuged and the DNA is captured by the membrane as the solution
passes through. (e) Wash buffers are added to the spin basket and (f) pass through the membrane
when centrifuged. (g) Typically 100
µ
l of elution buffer is added to the membrane; in the absence
of the chaotropic salt the DNA is released from the membrane and (h) is recovered upon a final
centrifugation
applied to the FTA
paper it is stable at room temperature for several years. Cellular
material lyses on contact with the FTA
paper and the DNA becomes bound to the
paper, which has been treated with chemicals to inhibit the growth of microorganisms
that might otherwise break down the DNA.
To analyse the DNA sample, the first step is to take a small region of the card,
normally a 2 mm diameter circle, place it into a 1.5 ml tube and the non-DNA com-
ponents are simply washed off, leaving only DNA on the card. The small circle
of FTA
paper is then added directly to a PCR (Figure 4.4). The FTA
paper
