Re cDNA synthesis

John Brennand, Tel 4639/5739/6636 John.J.C.Brennand at APVXC1.Pharmaceuticals.ZENECA.tmailuk.sprint.com
Tue Aug 8 07:02:46 EST 1995


Can't help you re. the number of mouse spinal cords you will need, but on the 
cDNA library front....

A couple of years ago we tested all the commercially available reverse 
transcriptases (AMV & MMLV) with control RNAs.  The winner by a country mile 
was Life Technologie's SuperescriptII - in terms of yield, product length and 
linkerability (is that a word ?).  I dont know if they do a complete synthesis 
and cloning kit - we usually put our own bits together for linkering and 
cloning.

Tip:   you must be very thourough in getting rid of excess linkers from the 
cDNA or they will kill you when you ligate in.  We would typically do at least 
2 spin column runs and an lmp agarose fractionation.  We found that the BstXI 
linkering into the commercially available BstXI cut plasmids followed by 
electroporation into XL1 blue, produced excellent libraries in terms of insert 
size, total numbers of recombinants and with a low % (<2%) of background 
clones

Good luck



More information about the Methods mailing list