I want to create an entry clone and I am using Invitrogen's BP Clonase Kit.
As per the manual, the Forward Primer with attB1 sites has two unspecified nucleotides as "NN".
Our lab uses "TA" in its position.
The manual has given an example with "CA".
The donor vector is pDONOR 201 with Spectinomycin resistant gene.
So my reaction is not working and I am not able to get any colonies after the transformation of the reaction. The use of "TA" works fine for other lab members.
Does anybody have any suggestions for an efficient BP reaction? Or its better to change primers?
It is different which nucleotides I have had at that position. I use the following databses to design my primers for Gateway and then just ajust them for matching annealing temperature.
ORF-Entry: http://worfdb.dfci.harvard.edu/index.php?page=searchwm
Promoter-Entry: http://worfdb.dfci.harvard.edu/promoteromedb/
This works just fine for me.
Besides that, when doing the BP reaction (and the LR reaction as well) I always incubate over night at 25 C. It gives higher yield in colonies.
Hope you get it to work.
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