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Digestion problem XbaI, methylation sensitive site


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#1 wincel

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Posted 24 September 2010 - 01:05 PM

Hi all, I have a very odd thing here and would like to know if anyone of you has another idea ...
I'm digesting (or try to) a plasmid with XbaI, it has been sequenced some time ago and should not be mutated. Digestion with another enzyme works fine. The XbaI tube is fresh as is the buffer and BSA.
The restriction site is overlapped by a dam site. Now I transformed the plasmid into SCS110 and it still does not digest.
I've done an actual calculation of the number of restriction sites per microgramm to make sure I have plenty of enzyme but to no avail.
I'm troubleshooting atm the bacteria (if they are SCS110 they should be Strep resistent) and try an overdigest with way more enzyme nevertheless.
Apart from that I am at a loss.
Any further ideas?

#2 Clare

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Posted 29 September 2010 - 01:29 AM

Hello :)

I just had the same problem a few weeks ago! I don't know what SCS110 is, but I just got some dam- competent cells from NEB and transformed my plasmid into them. After that, the XbaI site worked :)

Hope this helps!

Clare


Hi all, I have a very odd thing here and would like to know if anyone of you has another idea ...
I'm digesting (or try to) a plasmid with XbaI, it has been sequenced some time ago and should not be mutated. Digestion with another enzyme works fine. The XbaI tube is fresh as is the buffer and BSA.
The restriction site is overlapped by a dam site. Now I transformed the plasmid into SCS110 and it still does not digest.
I've done an actual calculation of the number of restriction sites per microgramm to make sure I have plenty of enzyme but to no avail.
I'm troubleshooting atm the bacteria (if they are SCS110 they should be Strep resistent) and try an overdigest with way more enzyme nevertheless.
Apart from that I am at a loss.
Any further ideas?



#3 phage434

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Posted 29 September 2010 - 04:02 AM

You should test your strain for XbaI protection. You can do this easily by isolating genomic DNA and testing to see if it can be cut with XbaI. If it remains uncut, you have a problem, potentially. Do this with (a different sample) cut with EcoRI, e.g., so you can see what cut genomic DNA looks like. Load a lane with no cutting.

#4 wincel

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Posted 13 October 2010 - 02:42 PM

Thanks for the reply. SCS110 is a dam/dcm negative bacteria strain from Stratagene. Testing the genomic DNA is an idea I haven't had yet, thanks phage434. I've meanwhile went with another design that excludes the XbaI site and worked well. But it is a good idea to check my SCS110 just in case I run into a similar problem in future.




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