PCR-vector-sequencing

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Dec 3 07:56:00 EST 1992


In article <2DEC92.22033639 at wums.wustl.edu>, wetsel_r at wums.wustl.edu writes...
>In a previous article, DMEIER at mis.mcw.edu wrote:
>>Hello to all,
>>  I am designing a set of primers which will give me a PCR product of
>>approx. 600 bases.  These primers will have EcoR I and Hind III 
>>restriction sites (one only for each primer of course).
>>  Is there a plasmid of choice I should put the PCR product in for 
>>sequencing?
>>  All replies will be gratefully received.
>> 
>>Daniel A. Meier
>>DMEIER at mis.mcw.edu
>----------
>Yes Dan....  Take your pick from the plethora of available cloning vectors. 
>Personally, I'd take the old reliable p-bluescript.  1) it has both sites 
>that you are using in its polyliker, 2) it has multiple primers for 
>sequencing, 3) in HB101, JM109, and Sure (Stratagene) cells it is present 
>in very high copy number, which translates into mgs/liter of t-broth or 
>L-Broth, and 4) if sequencing is difficult for some reason, one can roll 
>bluescript into a single stranded form for easier sequencing.
> 
>thats my $0.02 worth...
> 
>Best of luck,
>David
> 
Dan,
	We like the even older and still reliable M13 vectors. Rather
than fooling with sequencing off double stranded templates, which can
be difficult for some, the single stranded templates are much easier
to deal with.  As for putting two different restriction sites on either
end, that's really not necessary and also can be troublesome because
you'll need vector cut with both restriction enzymes.  Cutting with two
different enzymes usually gives lower background from vector self ligation
but this too may not be that useful since you are only trying to clone
a single, unique fragment and so what if you get some blue plaques?

thats my $0.02 worth...

Cheers.....bruce
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