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New ordered Restriction Enzymes not working, need help!


Best Answer bob1, 23 October 2013 - 01:41 PM

Fermentas/thermo fisher have a double digest compatibility program on their site that says that for NsiI and NdeI to use fermentas buffer R and a 2 fold excess of NdeI.  You may need to use more of the NdeI than you thought or do sequential digests in the respective buffers.

 

The single digests work, so ordering more is not going to help - the one you have is not faulty!

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#1 Celeste

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Posted 23 October 2013 - 12:59 PM

We ordered NsiI FastDigest from Fermentas. It is supposed to share the buffer with NdeI which is from Biolabs. I tried using FastDigest Buffer which is the buffer that came in with NsiI and NEBuffer 3.1 which is the buffer for NdeI, none of them works at optimal condition. Only a small fraction of the template was cut by both of the enzyme...

 

I did single digests to check their activity and both of them worked fine in their separate buffer.

 

 

Anything else I could do other than ordering new RE? 


Edited by Celeste, 23 October 2013 - 01:30 PM.


#2 bob1

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Posted 23 October 2013 - 01:41 PM   Best Answer

Fermentas/thermo fisher have a double digest compatibility program on their site that says that for NsiI and NdeI to use fermentas buffer R and a 2 fold excess of NdeI.  You may need to use more of the NdeI than you thought or do sequential digests in the respective buffers.

 

The single digests work, so ordering more is not going to help - the one you have is not faulty!



#3 Celeste

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Posted 23 October 2013 - 02:48 PM

Fermentas/thermo fisher have a double digest compatibility program on their site that says that for NsiI and NdeI to use fermentas buffer R and a 2 fold excess of NdeI.  You may need to use more of the NdeI than you thought or do sequential digests in the respective buffers.

 

The single digests work, so ordering more is not going to help - the one you have is not faulty!

Thanks soo much! 






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