The PCR product will be ~600bp in length.
CLONING
We currently use two cloning technologies available from Invitrogen: T-A and directional topoisomerase-mediated cloning kits (catalog #K2040-10, K2400-20). The directional cloning kit is designed for Invitrogen’s Gateway system. We use both kits according to the manufacture’s instructions. If using Topo-cloning, do NOT gel purify PCR products – it reduces the efficiency of the Topo-reaction.
pENTR/D-Topo SP6 primer: CACC GATTTAGGTGACACTATAG
For convenient identification of clones containing the proper insert (20-100% for Topocloning), a HindIII site has been designed into the loop of the hairpin. A second HindIII site exists 5’ of U6 promoter. Digesting clones with HindIII releases a ~500bp fragment.
SP6-U6 promoter PCR product sequence (with out hairpin)*.
SP6—HindIII—BamHI—U6 promoter→
Lower case = plasmid or U6 Genbank sequence
Upper case = U6 promoter sequence not included in the Genbank sequence.
Note: “N”= ambiguous sequencing read.
*This sequence is subject to minor changes upon further sequence confirmation
Good Luck using short hairpin activated gene silencing (ie SHAGGING)!Please feel free to e-mail me questions concerning PCR-Shagging.
Patrick J. Paddison
Watson School of Biological Sciences
Cold Spring Harbor Lab
paddison@cshl.org
