Editing Blood RT-PCR (section)

== Extraction of DNA (Recommended Protocol) ==

using  Grade 903 and Ahlstrom Grade 226 

This protocol is recommended if you need a large amount of DNA. If your testing does not require a lot of DNA, then typically you can use one 3 mm punch and follow the protocol on page 1.

1) Punch 3 circles from the centre of a blood spot using a 3.0 mm paper punch into a sterile 1.5 mL microfuge tube. To prevent cross contamination between samples, punch a clean piece of filter paper 3 times after punching out each sample.

2) Wash the punched circles by adding 500 µL of sterile water to the tube and pulse vortex 3 times, for a total of 5 seconds. Please note: If the wash is pink, the blood sample was not completely dried. Discard these punches and thoroughly dry the remaining blood spots before re-punching the card and repeating step 2.

3) Using a sterile pipette remove excess wash water from the tube and gently squeeze the circles against the side of the tube to remove as much water as possible.

4) Add 100 µL of sterile water to the tube. Ensure the circles are completely immersed in the water by briefly pulse centrifuging the tube for 5 seconds.

5) Transfer the tube to a heating block at 95oC-100 oC for 15-30 minutes.

6) At the end of the incubation period remove the sample from the block and pulse vortex the sample approximately 60 times.

7) Briefly centrifuge to separate the paper punch from the eluant. The eluant now contains the extracted DNA.

8) Using a sterile pipette tip gently remove the spent blood spot circles from the tube and discard them into a biohazard container.

9) Store the eluted DNA at –20oC until required.
[[Category:Protocols]]