[Methods-and-reagents] Sequencing Difficulties

Wofo novalidaddress at nurfuerspam.de
Fri Jun 24 04:23:59 EST 2005


Dear Zhonglin,

seems that your sequencing rxn gets trapped at the hairpin. You might try 
to add denaturing stuff like betain, DMSO, dITP etc etc like we did in the 
good (?) old (!) days of manual sequencing.

Wo


>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
>

>Wolfgang Schechinger, Dr. rer. nat.

>Med Clinic I

>Endocrine Research Lab

Bochum University Hospital "Bergmannsheil"
- the world's first casualty hospital for miners and steel workes -
Germany

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