triparental conjgation prblm

[Shahram Mori]

Dear Netters
I have been trying to do a tri-parental mating between DH5a ( which carries
a cyanobacterial cosmid library; the vector is pDUCA7 ) and Cyanobacterium
Anabaena 7120. I am  following the following protocol
1- grow 7120 to high density
2-wash in media + 5% LB
3-resuspend in media (bg 11) + 5%LB
4- frozen cosmid bank was grown in LB containing 50ug Km and Cm
5-wash with bg 11 + 5% LB -> resuspend in Bg11 +LB
6- grow my shuttle E coli Am187 in LB + Ap Cm
7- Wash in LB/bg11 resuspend in Bg11 + LB 
8- Mix the Cosmid + shuttle E coli and 7120 in equal proportions
and placed 0.5 ml aliquots on nitrocellulose filters on Bg 11 plates for 2
days
9-Transferred the filters to Bg 11 + 30ug Neo/ml and looked for exconjugants.

Unfortunately this protocol was not very successful. I would very much
appreciate it if someone who has had experience with triparental matings could
tell what I was doing wrong .
Thanks a milion
Cheers
Shahram
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