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How to subclone FoxO fusion protein expression plasmid into pDsRed1N1 vector


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#1 nrl81182

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Posted 18 July 2014 - 10:05 AM

Hey all,

I am very new to cloning. I want to use the FoxO expression plasmids, pECE-HA-FoxO3a WT and pECE-HA-FoxO3aTM (Addgene) and subclone the FoxO3a coding sequences into the pDsRed1N1 vector? How does one do that? Thanks in advance

 

Map of plasmids:

http://www.staff.ncl...sRED-N1_map.pdf

https://www.addgene.org/1787/

https://www.addgene.org/1788/



#2 bob1

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Posted 18 July 2014 - 01:46 PM

From the addgene pages you can see that the FOXO3 insert has been cloned into pECE using HindIII and XbaI - so ideally you would be able to match those sites in the correct order to the multiple cloning site (MCS) on the plasmid you want to insert into.

 

Looking at the map for pDsRed, it seems that it doesn't have XbaI in the MCS (this could be because either it has more than one XbaI site in the plasmid or it isn't there at all).  So, the easiest option will be for you to choose a couple of restriction sites on DsRed and add these to the 5' end of some primers, then amplify the inserts off the pECE constructs using a proof-reading polymerase.

 

If you can, choose a pair of sites that are well used (HindIII and BamHI spring to mind) - they are commonly used and readily available.  Note that you may need to adjust the sequence by a base or two to get it in frame with the DsRed.

 

Get someone to check your primers and potential cloning before you order and start amplification.






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