>1- Maximum how many extra bases I can have before the ATG
>codon of my "gene"?
In the Baculovirus system the late promoter has the sequence of "ATAAG" with
transcription initiating usually from the T or second A. The optimum distance
between this sequence and the start codon appears to be approximately 70 bp,
but the distance does vary between 3 and greater than 100 bps. The maximum
amount of untranslated leader DNA that you can insert will really be dependent
on the transfer vector that you choose; however, I wouldn't count on the
distance being greater than 100 bp.
>2- It appears that I do not have a lot of desirable restriction
>sites around the beginning of the ORF, how am I going to get rid of the
To get rid of the unwanted leader DNA you could try nested deletions with
ExoIII/Mung Bean Nuclease Kits. These usually work well but be prepered to
screen alot of clones to find one with the proper size insert.
> 3- I am planning to use polyhedrin promoter based vectors. The
>kits that the companies sell are very expensive (almost $500 for 5
>transfections). Which one is the best choice? Is occ based selection too
While I haven't used them, I have heard the the kits from Pharmingen usually
work quite well. The Baculogold kit is suppose to be very good. It initially
got bad reviews because of some poor quality control (uncut viral DNA which
gave very high background of nonrecombinants). I believe that these problems
have been corrected. If you use a selection with occlusion minus, it is very
east to detect in a plaque assay.
West Henrietta, NY
EMail: Bart_Corsaro_at_USLRMG01 at internetmail.pr.cyanamid.com