direct sequencing (35S) from PCR product

c.guihal at rbge.org.uk c.guihal at rbge.org.uk
Tue Oct 1 08:52:53 EST 1996


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



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