Tail DNA for PCR (No Organic Solvents)

 

 

1. Obtain the last 2mm of tail and place directly into 200ul 1X PCR Buffer
with Nonionic Detergents (PBND) in a 1.5ml microfuge tube. (Tails can be
stored at frozen in PBS or PBND until use.)

2. Add 1.2 ul ProK solution (I han't seen any problem with up to 5 ul ProK
and tail degredation is slightly faster) to each sample and place in a 55oc
bath.

3. Incubate at 55oc with occasional vortexing until tissue is degraded (1-3
hours).

4. Heat samples to 95oc for 10 min. in PCR machine or by boiling to
inactivate ProK.

5. Add 2ul(note)  processed Tail DNA / 50ul PCR reaction.
   note: this volume will vary depending on your particular PCR primers,
   their Tm etc....

~undefined Adapted from : Perkin Elmer Cetus: Amplifications: Vol. #2 PEC 1989; pp1-3.

PCR Protocol

Note: Lysis buffer for tail prep is PCR compatible (not usually clean
enough for restriction digests).

Generic PCR Conditions
2 ul (variable) tail DNA : do not vortex, avoid sediment in tube.
2uM final Primers
0.025U/ul Taq
100-200 uM dNTP's
1X PEC PCR Buffer w/out MgCl2
1.5mM MgCl2
H2O to final total volume of 50ul

Reagents
1) (PBND)PCR Buffer w Nonionic Detergents :
For 500 ml final volume:50 mM KCl                               1.87 g KCl
10 mM Tris-HCl (pH 8.3)##                                           5 ml 1M Tris-HCl Stock
2.5 mM MgCl2- 6H2O                                                    255 mg MgCl2- 6H2O
0.1 mg/ml gelatin                                                          50 mg gelatin
0.45% v/v Nonidet P40 (NP40)                                     2.25 ml NP40 
0.45% v/v Tween 20                                                     2.25 ml Tween 20

                 q to 500 ml w ddiH2O and autoclave 
Store frozen in 5 ml aliquots

2) Proteinase K  :  Made fresh for each use !
   10 mg Proteinase K is dissolved in 1 ml ddiH2O @ 4oc. Filter sterilize.