Cycle Sequencing Problems

[Bruce Roe]

In article <1992Sep14.190403.7540 at ac.dal.ca>, batc at ac.dal.ca writes...
> 
>  Hi dear netters
>  I have figured out that there are some people who are working on cycle 
>sequencing. I 've tried some times and I had had one constant problem.
>Though I 've got really strong signal I 've got stops in all four lanes.
>I was trying to sequence PCR products using (end labeled) one of the PCR primers
>and the other was internal primer. I hane used the New England Biolabs'
>"CircumVent Thermal Cycle Sequencing Kit". Is there any one who has any idea
>why this happens and what the solution could be? I would appreciate any
>oppinion.
>Thank you in advance
>Costas Batargias

Hi,
	Your problems could be due to several factors:
1. Garbage template - try cycle sequencing off highly purified pUC or
			some other very good control DNA.
2. Letting the reactions sit at room temperature too long either before
	or after cycling.  One of my students recently obtained multiple
	stops in all 4 lanes similar to what you have just described and
	it turns out that he was pipetting with his tubes in a rack on the
	bench rather than in an ice/water bath, then waiting upwards to
	5 minutes before putting them in the PECetus9600.  After the
	cycling reactions were done he then had an additional 5-10
	minutes at room temp on the bench before pooling and EtOH pptn.
	The combination of all this time at room temp. produced gels
	with lots of "template-associated" compression due to the Taq
	polymerase activity which DOES CATALYZE REPLICATION, abite slowly,
	even at room temperature.  But the template's secondary structure
	is not melted out and thus the "template-associated" compression.
	SUGGESTION: to be extra carefull, pipet the cycle sequencing reactions
		    on ice (ice/water bath) and pool the reactions after
		    placing them back into the ice/water bath once cycling
		    is complete.  Bottom line is don't let the reactions
		    sit at room temperature either before or after cycling
		    as the enzymes do have activity at these lower temp's,
		    just enough to screw things up........sigh
	At the recent Hilton Head Human Genome meeting, Bill Studier
	described a new 6-mer based primer sequencing method that had
	as it's initial step a primer extension at aprox. 0 deg C.
	i.e. in an ice/water bath.........gives one pause to think...
Cheers....bruce
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -