robert at arbo.microbiol.uwa.oz.au
Wed Jun 17 20:05:55 EST 1992
In article <920617082956.2020021a at ntet.nctr.fda.gov> WMELCHIOR at NTET.NCTR.FDA.GOV (Bill Melchior, NCTR/FDA) writes:
>trying to sequence ds DNA using the Sequenase kit (USB).
>Her 1.9 kb insert is contained in an M13 plasmid vector. She is
>preparing the DNA using large scale plasmid preps (alkline lysis) and
>purifying plasmid DNA through CsCl gradients. She has also tried
>alkline lysis mini-preps and plasmid recovery using silica. Her problem
>is that all she gets on the sequencing gel is a large smear.
If the plasmid contains a f1 origin of replication (such
as the pGEM series for example, she could easily produce ssDNA, which in our
hands sequences quite nicely with the Sequenase kit. A word of warning about
superinfections: do not assume that any white colony is going to give you a
good superinfection (even if it has the insert in it - assuming you have white/
blue selection). It might pay you to follow the instruction in the lastest
Maniatis et al about assessing the yield of phage from different colonies.
The phage pellet size differences we have seen from various colonies of the
same transformation, was quite substantial. Also give some thought to the choice
of superinfecting phage, we've only had to use M13K07, but some other users
reported poor yields with this phage whilst having good success with R408.
I think it is a combination of host/phage/vector/insert which to some extent
determines the success. Than, so it seems there is also a bit of black magic !
Make sure the moon shines over your right shoulder when harvesting the phage,
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