How much leaf for PCR

ANMR1 at UORDB2.bitnet ANMR1 at UORDB2.bitnet
Tue Sep 8 08:12:00 EST 1992


We can routinly amplify by PCR DNA isolated from approximately 50-200
leaf cells (microscopic sectors). We first make protoplasts fom these
cells in mannitol using cellulase and pectinase. The protoplasts are
spun down and resuspended in the following buffer, heated to 55degreeC
for 1 hour, boiled for 5 mins, spun again and the supernatant (typically)
20-30 microliters) is kept frozen until PCR. For each PCR we use 5-10
microliters of that DNA.
Buffer:
50 mM KCl
        10 mM Tris.HCl, pH 8.3
        2.5 mM MgCl2
        0.45% NP-40
        0.45% Tween-20
        3microl/10 ml of Proteinase K (20 mg/ml stock, kept frozen at -20degreeC
) to be
added just before the experiment. (filter sterilized)

Let me know if you have any problem.
Animesh Ray



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