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Digest two sites located next to each other

XbaI salI digest neighbouring sit digest with no gap

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5 replies to this topic

#1 rathnasingh

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Posted 05 August 2014 - 01:01 AM

I have this sequence in a vector (tctagagtcgac). It contains XbaI (tctaga) and SalI (gtcgac) restriction sites located together without any gap. I would like to clone a DNA fragment between these two sites, XbaI and SalI. Is it possible to digest the vector DNA without any problem?

 

Thanks



#2 Trof

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Posted 05 August 2014 - 02:24 AM

Enzymes usualy need few bases on each side to cut. If one of the enzymes cuts its sequence, the other has no double strand overhang.

My best guess is, if you do a single digest the other enzyme will cut only at a very low frequency and if you do a double digest, the site would be randomly cut only by one or the other.


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#3 phage434

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Excellent

Posted 05 August 2014 - 05:35 AM

I agree. If you do want to try, digest with SalI first. It's a lot fussier than XbaI. Better would be to PCR the vector with the cut sites you'd like (not SalI) and sufficient overhang.



#4 rathnasingh

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Posted 05 August 2014 - 05:13 PM

I agree. If you do want to try, digest with SalI first. It's a lot fussier than XbaI. Better would be to PCR the vector with the cut sites you'd like (not SalI) and sufficient overhang.

Thanks. Let me try this cloning with SalI and followed by XbaI. According to NEB, both the enzymes could cut one or two bp overhang to certain percentage. Looks encouraging. 



#5 Rajpoot

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Posted 05 August 2014 - 06:45 PM

You can proceed with your cloning and hopefully there will be sufficient number of clones. Last week I did cloning into a vector bearing Nhe1  (GCTAGC)  and EcoR1 (GAATTC); sites  located very close  to each other. I did double digest overnight and faced no problem at all.

 

sequence on vector: ......GCTAGCGAATTC.....

 

Good luck!



#6 Rajpoot

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Posted 05 August 2014 - 06:51 PM

You can proceed with your cloning and hopefully there will be sufficient number of clones. Last week I did cloning into a vector bearing Nhe1  (GCTAGC)  and EcoR1 (GAATTC); sites  located very close  to each other. I did double digest overnight and faced no problem at all.

 

sequence on vector: ......GCTAGCGAATTC.....

 

Good luck!







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