What is the protocol for cloning your own hairpin design into the pGIPZ empty vector?

We do not have a protocol for cloning your own hairpin design directly into the empty pGIPZ vector. We used a cloning protocol that moved pre-existing hairpins from our pSM2 retroviral vector into the empty pGIPZ lentiviral vector, so we recommend that customers clone their hairpin design into the pSM2 empty vector according to the Paddison et al. (2004) "Cloning of short hairpin RNAs for gene knockdown in mammalian cells" reference and then use the cloning protocol in the pGIPZ product manual to move the hairpin from the pSM2 vector into the pGIPZ empty vector. There are a couple of differences between moving an already existing hairpin from the pSM2 vector into the pGIPZ empty vector versus cloning your own hairpin design into pSM2 empty vector and then moving the hairpin from pSM2 into the pGIPZ empty vector. Subcloning a hairpin that was ALREADY in the pSM2 vector will include a unique 60 nt barcode. According to the pGIPZ cloning protocol, the XhoI – MluI fragment size that you will be moving from pSM2 to pGIPZ will be 345bp. Upon PCR to confirm the cloning, the band sizes that you should see if shRNA was successfully inserted versus the shRNA not being inserted is as follows: shRNA inserted = 603bp No shRNA inserted = 516bp When you clone your own hairpin design into the pSM2 empty vector it will not have a barcode. The XhoI – MluI fragment size will be approximately 280bp. Upon PCR to confirm the cloning, the band sizes that you should see if shRNA was successfully inserted versus the shRNA not being inserted is as follows: shRNA inserted = 543bp No shRNA inserted = 516bp The differences between these two will be too small to resolve on a gel. Instead of running PCR products on a gel, you will have to sequence the clones to confirm whether or not she you were successful in your cloning. To have an idea of how many colonies will have to be sequenced in order to obtain a correct inserted shRNA, it is important to perform a "no insert control" ligation with the pGIPZ empty vector. The level of background (no insert present) will be based on the number of colonies seen on the "no insert control" plate versus the "insert added" plate. For example, if 3 colonies were observed on the "no insert control" plate and 30 colonies were observed on the "insert added" plate, the odds are very good that the ligation of a hairpin into the vector was successful. However, if 9 colonies were observed on the "no insert control" plate and only 10 colonies were observed on the "insert added" plate, the odds are not very good that the ligation of a hairpin into the vector was successful. ** Please note the pSM2 and pSMP libraries, constructs, gene sets and families and RNAintro kits have been discontinued.