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
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___     Department of Medical Microbiology																		___
___     Imperial College School of Medicine at St Mary's    ___
___     London W2 1PG																																		     ___
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