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The sequencing facility

Protocol BigDye v.3.1

1. Quantifying DNA

                     

TemplateQuantity
PCR product: 
100-200 bp  1-3 ng
200-500 bp3-10 ng
500-1000 bp         5-20 ng
1000-2000 bp       10-40 ng
>2000 bp       20-50 ng
  
Single-stranded        25-50 ng
Double-stranded   150-300 ng
Cosmid, BAC
     0,5-1.0 µg
Bacterial genomic DNA           2-3 µg

 

2. Preparing sequencing reaction

Reagent      Quantity
Big-Dye version 3.11 µl
Sequencing buffer (store at 4C)1 µl
Template (see table above)200 ng
Primer   3,2 pmol
Deionised water    q.s.p. 10 µl 
 Total 10 µl



N.B. Alternatively, use up to 1,5 µl of the sequencing buffer and 5 pmol of primer 

 

3. Performing cycle sequencing in a  thermal cycler


1                          96º C, 5 min

2      25 cycles     96 º C, 10 sec.
                            50 º C, 5 sec.  *
                            60 º C, 4 min.

3                          4 º C

* Annealing temp should be optimized according to the primer used (typicallY Tm-5)


N.B. Add 10 µl deionised water after the sequencing reaction to deliver your sample