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oligo hybridization and ligation


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#1 Petra Kowalski

Petra Kowalski

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Posted 18 May 2002 - 10:04 AM

Hello everybody,

I want to clone a ribozyme (oligo 40-mer) into a tet-response vector. The vector is cut blunt end, so I need to blunt end ligate the oligo into it. But, my first 24 clones revealed all the empty vector. The ratio between vector and ribozyme  oligo in the ligation mix was 1:5000, so much more insert than actually required. Additionally, I added PEG in the mix, in order to bring the molecules together. If anybody has ideas about blunt end ligation of short oligos and vector, I would like to hear about it.
The second problem might be that the previous hybridization did not work. I usually do it 2min at95, 8min at 60 and on ice after that. Has anybody a better hybridization protocol. AND: Is there a possibility to proof whether hybridization took place or not. In other words, can I determine somehow, if an oligo is single stranded or double stranded DNA??? Of course, it should not take 1 month.
All ideas and suggestions are appreciated very much!
Thanks in advance.
Petra


#2 Sam Brilliant

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Posted 18 May 2002 - 11:35 AM

This ribozyme ligation will work only with a 12 hour incubation with ligase at 4degrees Celsius.


Sam Brilliant
Ph.D. Rescue Team
A Division of DSTF-Global
http://www.dstf.bigstep.com





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