designing shRNA constructs - (Apr/07/2008 )
hi all,
i've recently purchased a HuSH shRNA gene silencing set from origene. within this set come an empty vector which the company say they allow you to use if you want to insert your own shRNA constructs... my question is, if i know the sequence i need to insert (sequence - loop - reverse sequence - termination sequence) how do i go about getting this into my empty vector? does it have to be a double stranded oligo which i use restriction enzymes to insert?
any help is much appreciated
adios
-flashboy-
QUOTE (flashboy @ Apr 7 2008, 07:23 AM)
hi all,
i've recently purchased a HuSH shRNA gene silencing set from origene. within this set come an empty vector which the company say they allow you to use if you want to insert your own shRNA constructs... my question is, if i know the sequence i need to insert (sequence - loop - reverse sequence - termination sequence) how do i go about getting this into my empty vector? does it have to be a double stranded oligo which i use restriction enzymes to insert?
any help is much appreciated
adios
i've recently purchased a HuSH shRNA gene silencing set from origene. within this set come an empty vector which the company say they allow you to use if you want to insert your own shRNA constructs... my question is, if i know the sequence i need to insert (sequence - loop - reverse sequence - termination sequence) how do i go about getting this into my empty vector? does it have to be a double stranded oligo which i use restriction enzymes to insert?
any help is much appreciated
adios
You have to design shRNA and order them from the company which provides primers to your lab. You will have a top and bottom strand. Anneal them and ligate it into the vector provided.
-scolix-
cheers scolix... i've just read a paper which says that this is an unreliable way of doing it, and using one strand as a template and Phi29 polymerase to produce a complimentary strand is better???
-flashboy-