Long-Range DNA-sequencing

Victor Levenson U10784 at UICVM
Mon May 15 14:51:07 EST 1995


Sequencing folks,
For the last couple of years or so I was quite happy with cycle-sequencing kit
from Epicentre Technologies. I use 32P and run longer 5% gels to resolve more
bases. In the original kit, I use a little bit more primer per reaction than
required by protocol (2.5 pmoles instead of 1.5) and no more than 20-40 ng of
plasmid DNA. Epicentre also have long range termination mix of ddNTPs. PCR
conditions with most 20-mer primers: 1 min 95oC, and then 35 cycles of 1 sec
95oC, 1 sec 60oC and 30 sec 72oC. It actually works even on colonies.

Alexander Kolchinsky
kolchins at uicvm.uic.edu



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