Q: Trouble with Kindergarden Ligation (SspBI/ApaI)
Koen De Smet
k.desmet at nospam.ic.ac.uk
Mon Sep 22 07:04:18 EST 1997
Wolfgang Schechinger wrote:
> Hi you all molbio gods and experts!
Don't call me God or expert, because I don'thave the answer. But we have
had the problem! Maybe these particular enzymes need more than 4 extra
bases to work properly? From experience we know that 2 extras are
enough for BamHI, but at least 4 are needed for HindIII.
Since you use Pfu, the PCR product should be blunt. You can ligate this
(without digest) in pUC or so, and then cut out the insert. That is how
I once got around it.
(I assume you have double- and triple checked the sequecne of your
primers! Taht is apparently also a common problem!)
> !Junk mail is *not* appreciated!
> SPAMs will be answered automatically by sending my 128MByte virtual memory file.
> This is *NO* joke.
BTW, One reason why one tends to receive spam is because companies use
software to compose lists of email addresses on newsgroups. So set up
your browser so that there are some extra letters in your email address.
In your signature, specifiy which have to be deleted for reply. The spam
software doesn't read the signature, so the spammers email list contains
the disfunctional email address. You won't get bothered, and they will
eventually get an error message. They only expect 0.1% of spam messages
to give a results (a sale), but they may hopefully think twice if the
vast majority of replies are error messages.
Koen De Smet
To reply by email, remove "nospam." from the above address
___ Department of Medical Microbiology ___
___ Imperial College School of Medicine at St Mary's ___
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