I'm trying to build a construct which adds a 3X HA tag to the N-terminus of the protein and this construct is going to be expressed in transgenic flies. I don't know how exactly to do that! each HA is 27 nucleotide long so I probably can't include all (81 N) in one primer? also it's too small to be seprately cloned into my vector? does any body know how I should proceed?
Thanks
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#2
bob1
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Posted 23 July 2011 - 08:48 PM
You can definitely add it to a primer. It also isn't too small to be able to be cloned separately if you can find a source for the HA - people add linkers of 8-10 bp to sequences frequently.
#3
Opal
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Posted 24 July 2011 - 07:04 AM
well I have a source for HA but I don't know how to amplify just an 81 n fragment how to then purify and clone it into a 9 kb vector !!! it probably needs a special procedure!
#4
bob1
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Posted 24 July 2011 - 03:58 PM
Not at all, just find out what the restriction sites on either side of the HA are (and on the plasmid you are cloning into), cut and purify, then clone as you would normally.
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Posted 24 July 2011 - 06:56 PM
and does anybody know how many HA do we need to add so that it could be detected in a western blot by the anti-HA? cause if only one is enough my life will be much easier.
#6
bob1
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Posted 25 July 2011 - 09:53 PM
One is enough (I have done this), though the signal will be stronger if you use 3x.
