AFLP troubleshooting

Bodychecker megacheck at hotmail.com
Wed May 16 13:02:04 EST 2001


hi,

usually more Taq helps (2.5 U/ 50 mikroliter). most buffers are simple KCl
buffers. Try the Qiagen buffer, it is different. Also much longer
Annealing/extension times help a lot.

Good Amplifications!
Bodychecker

"Tim Jackson" <tim.jackson at nrc.ca> schrieb im Newsbeitrag
news:3B02ACB7.FA09B3E5 at nrc.ca...
> i'm using the GibcoBRL AFLP kit on Atlantic halibut.  i'm visualising my
> product on an ABI 377 (fluorescent labelled EcoRI primers).  mostly
> things are working very well except that my signal intesity for bands >
> 350 bp drops way off (i assume because of decreased amt. of PCR product
> for the larger fragments).  i tried a few different Taq's and settled on
> one which helped improve my signal from 200 to 350 bp but haven't been
> able to make any improvements beyond that.  i thought i might try
> increasing my extension time (currently 94 for 30s, 56 for 30s and 72
> for 60s).
> anyone have any bright ideas???
>
> thanks!
>
> tim
>





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