triparental conjgation prblm

Leonard N. Bloksberg bloksber at pilot.msu.edu
Wed Jan 26 10:11:00 EST 1994


In Article <2i29giINN176 at dns1.NMSU.Edu> "smori at nmsu.edu (Shahram Mori)" says:
> 
> 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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> 
> 				
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> 
Hi Sharam
	My favorite protocol for tri-parentals is to use 1 part helper
strain : 2 parts donor strain : 5 parts recipient strain.  Swirl the mix
of all 3 strains in a tube breifly and filter the mess down onto a 47mm, 
.45u filter.  Place the filter on a plate of media which favors the 
recipient strain, with no antibiotics overnight, or until a thick slime
grows on the filter.  Vortex the filter into fresh medium and plate on
selective medium.  You can fudge the proportions, but 1:1:1 never gave me
anything either.
	For a different approach, a buddy of mine tried to do complementation
cloning by mating the cosmid library back in from E. coli, and never got
it to work.  He finally tried electroporating the library in instead, and
had his clone in a few weeks.  You may want to keep this alternate approach
in mind.  
	Good Luck
.
.
	Leonard N. Bloksberg
	bloksber at pilot.msu.edu
.
.
.



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