single cell PCR
s6311190 at ms22.hinet.net
Thu Jun 14 10:56:17 EST 2001
I am trying to run single cell PCR. Now I encounter a problem. After the
second PCR, there was no product.
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
Pre-PCR 94oC, 5 min
PCR 94oC, 1 min
55oC, 1 min
72oC, 1 min
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
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