Dear manual sequencing user,
I have used the Amersham kit for a long time now, and this has always
worked well. If you can not afford an automatic sequencer, this is a
very good method especially for short target DNA (400bp).
I used the enzyme pretreatment included in the kit, and 35S as
radiolabel.
So I sequence directly from my PCR products. As long as you check on a
agarose gel that your product contain only one target (one band), you
should not have any trouble.
I usually run the reactions twice during different time (2hours/5hours)
that let me read up to 350bp.
I suggest that you use two primers to start from both strand. Then you
may choose to use two more to complete and confirm your sequences.
I hope this will help.
C.GUIHAL
Caroline GUIHAL
Scientific and technical services Department
Royal Botanic Garden Edinburgh
20A inverleith Row
Edinburgh EH3 5LR
Scotland, UK
Tel: 0131 552 7171
Fax: 0131 552 0382
e.mail: C.GUIHAL at rbge.org.uk
