[Methods-and-reagents] Sequencing Difficulties

Zhonglin Chai Zhonglin.Chai at baker.edu.au
Thu Jun 23 22:23:55 EST 2005


Dear Netters,

We have had difficulties to sequence a few DNA constructs. These constructs are designed to express hair-pin structured siRNA. A proportion of the DNA (about 50 bp) may fold and hybridize to itself after denatured (a single stranded). The sequencing reads ok (but short) until it reaches the siRNA region where it suddenly loses signal (no signal at all). It looks like a sequencing of short PCR product template (ours are circular plasmids). We have tried to simply increase the temperatures for 96oC, 58oC and 72oC steps, but still have the same problem.

Do you know why, and how to solve the problem?

Regards.

Dr Zhonglin Chai, PhD 
Diabetic Complication Group 
Baker Heart Research Institute 
Commercial Rd, MELBOURNE, VIC 3004
AUSTRALIA



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