a followup on this. you might have seen this (http://dx.doi.org/10.1016/j.celrep.2013.11.020). There are improvements to these miR-based shRNAs. These would necessitate altering the vector a bit.
actually am a bit confused with the restriction site... do i have to clone it b/w the EcoR1 and Xho1 site??
No, Actually I use XhoI and Mlu I sites to clone the shRNA. Its clearly mentioned in the manual how to do this. Please find attached below. page No.7
firstly,thank you very much for the information..i saw this in the manual before but could not correlate the sites with the shRNAmir sequences (i have a clone with shRNAmir already inserted in TRIPZ vector from open biosystems; so i know the shRNAmir sequence)....
Your cloning with XhoI and MluI was succesful,right? You got good knockdown of your gene?
And did you get any flanking sequence to be used for the shRNA desgn from their site?
Ok, so please find attached the sequence file of the TRIPz vector with a non silencing insert in it. If you carefully see, the scrambled shRNA is cloned between the XhoI and Mlu I. Yes it worked for me. However I do not understand why you want to clone it in the TRIPz vector if you have already in it. Did I get you correct?
PS: You can open this file in any sequencing analysis software, if nothing works download this software, its a free plasmid editor
http://biologylabs.utah.edu/jorgensen/wayned/ape/
Thank u once again
no, i want to clone shRNA of a different gene..
& can you give me some idea about the flanking sequence of shRNAmir that you designed?
Unfortunately I did not design an shRNAmir. I amplfiied or restricted from another vector and cloned into the TRIPz
Just a note: if you clone the shRNA between EcoRI and XhoI you will leave a bar-code intact. If you replace with XhoI and MluI you will be removing the bar code portion but more importantly you will take our half of the miRNA.
We have been making shRNAs compatible for pTripZ and pGipZ in a number of ways. Often you can pull these sequences directly for your gene from openbiosystems. Otherwise I think our lab uses online software to create new shRNAs.
Hello people I am also facing the same issue in cloning my shRNA in to TRIPZ .I used xhoI and MLu1 as my RE sites .Despite of getting the colonies on my transformed plates and no colony on ctr plate ,I still didnot get success ,as verified by my sequencing results.
Do you have any suggestions to mend the bridges..
Hi Kusum, that sounds like more of a general cloning issue. What do you see when you sequence? Still an empty vector? Also, how did you design the shRNA? For what it's worth, I found this to be helpful for designing my shRNA to put into the TRIPZ vector:
http://cshprotocols.cshlp.org/content/2013/7/pdb.prot075853.abstract
-Luke
a followup on this. you might have seen this (http://dx.doi.org/10.1016/j.celrep.2013.11.020). There are improvements to these miR-based shRNAs. These would necessitate altering the vector a bit.
Follow up. I just recently cloned an shRNA construct into the empty pTRIPZ vector. I designed a mir-30 based shRNA construct following the protocol from G. Hannon's lab (see publications) and cloned into the XhoI/EcoRI sites. I was able to get knockdown of my target gene.
Article Cloning of short hairpin RNAs for gene knockdown in mammalian cells
Article Creating an miR30-based shRNA vector
Hi!
Do you know if it is possible to clone a microRNA into the pTRIPZ vector instead of a shRNA?
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