cycle sequencing reaction questions

[Silke Beismann]

WZhao68 wrote:
> 
> Hello there,
>        Does anybody have a good, robust dsDNA cycle seqencing protocol other
> than the Perkin Elmer one. Somebody
>        use DMSO in the reaction, what does it do? Do you have to increase the
> amount of DNA when the plasmid is big?
>        Does high salt concentratin in DNA sample inhibit the cycle sequencing
> reaction?
>        Thanks in advance!
>        David

Hi David,
we use (successfully) the following protocoll in our lab:



· Master-Mix for every sequence:
4 × 50 ng/kb	plasmid	
4 ×  2 pmol	primer	
4 × ad 6 µl	H2O

· Make aliquots of 6 µl each

· add 2 µl Reactionsmix (e. g. Amersham)

· Cycle-Sequencing:
2'	98 °C \
15"	95 °C  |
20"	55 °C  | 25 ×
30"	70 °C /

15"	95 °C  |
20"	70 °C  | 10 ×

· Shortly bevor loading the gel: 
heat samples 2' 95 °C 
add 8 µl formamide marker-solution 


As you can see the amount of dna used depends on the size of the plasmid
(5 ng for each kb plasmid).
We add DMSO usually to PCR samples. It helps denaturating the dna,
especially if there is extensive secondary structure. In cycle
sequencing additin of DMSO seems to be unneccessary.

Since I do not know the effect of salt in cycle sequencing I cannot
answer your last question. 
I hope the other suggestions help!

Good luck,
Silke