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Molecular biology

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Molecular biology

Postby maheshgen » Mon Mar 14, 2011 1:48 pm

I have a problem in restricting pET-28a(+) and pET-28b(+) vectors with NcoI and XhoI. I have tried Fermentas enzymes with buffer Tango. I tried many times but went invein. Can anybody guide me in this as I am in desperate need of this vector to proceed my work. Thank you in advance for the advice
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Postby JackBean » Mon Mar 14, 2011 8:27 pm

they seem fine, both Fermentas and NEB Enzymes show they should work fine in wide range of buffers, so that shouldn't be a problem. What about other conditions? How do you check the result? Where did you get the plasmid?
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Re:

Postby maheshgen » Mon Mar 14, 2011 9:22 pm

JackBean wrote:they seem fine, both Fermentas and NEB Enzymes show they should work fine in wide range of buffers, so that shouldn't be a problem. What about other conditions? How do you check the result? Where did you get the plasmid?



Thanks jackBean for reply. The enzymes are working. We have pET-28 a-c (+) vectors in our lab. I tried to restrict with XhoI and NcoI seperately but nothing seen on gel. I tried even EcoRI, BamHI, and HindIII but didnt restrict. I dont understand where is the problem. I tried to isolate fresh plasmid but no results. I even tried different concentrations of buffer and enzyme. But before i didnt experience such problems.
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Postby JackBean » Tue Mar 15, 2011 9:50 am

seems rather like problem with the vector. How did you check you have really the vector you want? How was your DNA purification?
Are you sure the vector is as from manufacturer, wasn't it cut already in the past?
http://www.biolib.cz/en/main/

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Re:

Postby maheshgen » Tue Mar 15, 2011 10:51 am

JackBean wrote:seems rather like problem with the vector. How did you check you have really the vector you want? How was your DNA purification?
Are you sure the vector is as from manufacturer, wasn't it cut already in the past?



we use these vectors and they all function well. Even i cut these before without any difficulty. But now something strange is happening. I put these vectors side by side with pUC19 plasmid and found that its very bigger on the gel compared to pUC19.

mahesh, UK, Bratislava
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Postby JackBean » Tue Mar 15, 2011 11:49 am

UK, Bratislava? :lol: To zní zajímavě :)

Then you should find out, what's going on, you probalby have the vector with some insert, probably in place of your restriction sites
http://www.biolib.cz/en/main/

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Postby RichardvanLeeuwen » Fri Apr 01, 2011 12:30 pm

I had the same problem with digestion of pET28a(+) with BamHI/XhoI. Also I used Fermentas RE and obtained more bands then expected. By using the FastDigest RE from Fermentas I was able to obtain the correct bands.
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