DNAFromWholeBloodforPCR

(fromHiguchi,R.(1989)Amplifications2:1-3)

1. Obtain65-100µlofbloodbyretro-orbitalbleedwithaheparinizedmicrocapillarytube.Expelbloodimmediatelyintoa1.5mlmicrofugetubecontaining20µlof10mMEDTA.Miximmediatelytopreventclotformation.Storeoniceuntilprocessing.

2. Add200µlLysisBuffertoeachtubeandvortextosUSPendevenly.

3. Microfuge25secondsat16000xgtopelletnuclei.

4. Removeanddiscardsupernatantandrepeatsteps2-4twomoretimes,oruntilnohemoglobinremains.

5. Resuspendnuclearpelletin100µlPBNDwith60µg/mlproteinaseKandincubateat55Cfor60minutes(orovernight,ifconvenient).

6. Heatsamplesto97Cfor10minutestoinactivateproteinaseK.

7. Add1-5µlofDNAsolutionfora25µlPCRreaction.

Reagents:

1)LysisBuffer

0.32MSucrose10mMTris-HCl(pH7.5)5mMMgCl21%v/vTritonX-100

2)PBND(PCRBufferwithNonionicDetergents)*

50mMKCl10mMTris-HCl(pH8.3)2.5mMMgCl20.1mg/mlgelatin0.45%(v/v)NonidetP400.45%(v/v)Tween20

AutoclavetosterilizeanddissolvegelatinStorefrozen

*AddproteinaseK(60µg/ml)immediatelypriortouse)

Typical25µlPCRreaction:

1-5µlDNA2.5µl10xPerkinElmerbuffer,1.5mMMgCl2(final)2µl2.5mMdNTPmixture(2.5mMeachdNTP,200µMfinal)0.5µl20µMforwardprimer(0.4µMfinal)0.5µl20µMreverseprimer(0.4µMfinal)0.1µlTaqDNApolymerase(candecreaseto0.05µl)dH2Oto25µl

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