Which primers are better for colony PCR? - (Oct/10/2006 )
Hi,
I am going to amplify the promoter of a gene by PCR and clone it into TOPO TA cloning vecter.
I want to select correct colonies by colony PCR.
I woud be happy if anyone could tell which primers to be used, inside or outside the
insert.
Thank you.
-slab-
Hi Slab,
I always try and use a primer from within the insert and one corresponding to the plasmid, it gives the most information ie. presence of insert, direction of insert, number of inserts. If you use two primers from the vector it only tells you whether there is an insert and what size it is not what it is or anything about it.
Scott
-Scott-
We use sucessful M13 primers (for pbluescript vector)
-hobglobin-
QUOTE (Scott @ Oct 10 2006, 11:17 PM)
Hi Slab,
I always try and use a primer from within the insert and one corresponding to the plasmid, it gives the most information ie. presence of insert, direction of insert, number of inserts. If you use two primers from the vector it only tells you whether there is an insert and what size it is not what it is or anything about it.
Scott
I always try and use a primer from within the insert and one corresponding to the plasmid, it gives the most information ie. presence of insert, direction of insert, number of inserts. If you use two primers from the vector it only tells you whether there is an insert and what size it is not what it is or anything about it.
Scott
And if you use 2 primers of the insert its highly likely to get a false positve by Cpcr, so always use 2 different primers. see attachment
-rajgene-
thanks for that interesting article
-perneseblue-