single cell PCR

water s6311190 at ms22.hinet.net
Thu Jun 14 10:56:17 EST 2001


Hi,
I am trying to run single cell PCR. Now I encounter a problem. After the
second PCR, there was no product.

PCR condition:
First, I put a cell into a 0.2 ml PCR tube with 5 ul alkaline lysis
buffer (50 mM DTT, 200 mM KOH). Then incubate it at 650C for 5 min.
After incubation, 5 ul neutralization buffer (900 mM Tris-HCl, 300 mM
KCL, 200 mM HCl) was added to the mixture. Then the mixture was brought
to a final volume of 50 ul (10 mM Tris-HCl, 50mM KCl, 1.5mM MgCl2, 0.25
mM each of the four dNTPs, 0.5U/ul Taq DNA polymerase, 1uM each of
primers).

Pre-PCR  94oC, 5 min

PCR     94oC, 1 min
            55oC, 1 min
            72oC, 1 min
           33 cycle
Post-PCR  72oC, 5 min

Then 2 ul fist PCR product was used as template for same PCR. The
condition was as above including primers.

The PCR condition described above works fine with genomic DNA.
Could anyone make a suggestion to me.

Thank you for help!

Sincerely, Chang Ya-Hui
2001/6/14


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