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TAIL PCR ANALYSIS
For this service Investigators have three
purification options. Our standard purification protocol from the Jackson
laboratory, BioPioneer Quick Extraction kit (available in the facility)
or your very own custom protocol.
Jackson Laboratory protocol
1. Obtain the last 2mm
of tail and place directly into 200ul 1xPCR 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 1-2ul ProK
solution (Made fresh for each use! 10mg Protenase K is dissolved in 1ml
dH2O @4ºC.Filter,sterilize.)
3. Incubate at 55ºC
with occasional vortexing until tissue is degraded or O/N is o.k.
4. Heat samples to
95ºC for 10min. in PCR machine or by boiling to inactivate ProK.Put on
ice to cool down.
5. Spin lysate at
max.speed for 2min.Transfer sup into a new tube.
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
2) q to 500 ml w
ddiH2O and autoclave Store frozen in 5 ml aliquots
3) Proteinase K
: Made fresh for each use ! 10 mg Proteinase K is dissolved in 1 ml
ddiH2O @ 4oc. Filter sterilize
Jackson
Laboratory
Optimizing PCR protocol
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