Monday, 19 November 2007

DNA protocol (2)

Isolation of DNA from buccal swabs
50 mM NaOH
1M Tris.Cl, pH 6.5
70% ethanol
CYTo-Pak with Cyto-soft brush (CP-5B; Medical packaging) or Caliber-Cotton-Tipped Apllicator swab (size 6 in; Allegiance Healthcare)
95 degree heating block


praspowt said...

1. Having the subject lightly rinse out his or her mouth with water twice. Remove a brush or swab from its sleeve, avoiding contamination as much as possible, and roll it against the inside of the subject's check for 3 sec. Place the brush back into the sleeve. Store up to 1 week at 4 degree celcius, but extract as soon as possible
The yield of DNA from buccal epithelial cells can be highly variable because of variations in both the subject and the technique of the collector. It is wise when possible to obtain two brushes or two to four swabs. Care should be taken not to roll or brush too hard, as repeated hard strokes can cause abrasion, soreness, or even bleeding.

praspowt said...

2. Pipet 600 microlitre of 50 mM of NaOH into a 1.5 ml microcentrifuge tube. Cut off the sample- containing end of the collection brush or swab using scissors and place it in the microcentrifuge tube using sterile forceps (two swab tips may be plced in one tube). Sterilize scissors both before and after use by washing in 70% ethanol.

praspowt said...

3. Vigorously vortex the tube and place in a heating block at 95 degree celcius for 5 min to complete cell lysis. Remove the brush or swabs from the microcentrifuge tube using sterile forceps, leaving behind the residual solution containing the DNA. Neutralize the solution by adding 60 microlitre of 1 M Tris.Cl, pH 6.5, and vortex the tube again.
DNA is now ready for quantitation, or it can be used directly for PCR. Buccal DNA samples to be used for PCR may be stored at either 4 degree celcius or -20 degree celcius.
Since the DNA is not highly purified, exact quantitation is difficult and usually not necessary. typically 5 to 10 microlitre of the DNA solution is suffficient for a successful PCR reaction. Estimated of yield can be made using the DNA dipstick kit (Invitrogen).
DNA yields are highly variable, but 4 to 8 microgram/brush is typical.