Protocol BigDye v.3.1

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1. Quantifying DNA

Template	Quantity
PCR product:
100-200 bp	1-3 ng
200-500 bp	3-10 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 3.1	1 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

13.01.2015