Do you need supercoiled pDNA for ds-sequencing?

Jim Owens jow at helix.nih.gov
Wed Nov 2 08:27:50 EST 1994


In article <rcwieg-0111941657260001 at rcwieg.monsanto.com> Roger Wiegand,
rcwieg at ccmail.monsanto.com writes:
>In article <MURIANAP.30.000C1D5C at FOODSCI.PURDUE.EDU>,
>MURIANAP at FOODSCI.PURDUE.EDU (Pete Muriana) wrote:
>
stuff deleted
>> Now the question - the DNA sequencing facility representative
indicated that 
>> we need to have **supercoiled** plasmid DNA to sequence from.  Is this
true? 
>> and why wouldn't a linear double-stranded fragment work? Am I missing 
>> something here?
>> 
>
>This is nonsense. Both cycle sequencing and Sequenase work fine from
>linear DNA such as PCR products. We have a number of examples where
>nicking or linearizing (either mechanically or using enzymes) plasmids
>results in a 5-10X *increase* in signal strength for cycle sequencing.

It seem to be a common phenomenon that some labs can get one technique to
work while other labs cannot.  Roger gets better results with nicked or
linearized plasmid DNA, I do not.  

Assuming that your sequencing facility will be doing the sequencing
reactions for you, Pete, I'd suggest you give them what they want since
that works for them.  If you will be doing the sequencing reactions, try
out both methods and see which works better in your hands.

By the way, Pete, your e-mail box is full, ten thousand messages,
according to your daemon.  I guess that mean you do not use the address
above for mail?

Good luck,

Jim Owens



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