This kit supplies reagents sufficient to make 5 adenovirus vectors, each capable of silencing the expression of a target gene.  An insert that includes a sequence from the target gene is ligated into the pQuiet-U6 plasmid.  The insert is constructed by annealing compementary oligonucleotides to one another.  Each oligonucleotide is designed with a 4-base overhang at its 5'-end.  The overhangs complement vector sequences that remain after it has been digested with a pair of restriction enzymes (Spe I and Mlu I).  After confirming that the correct sequence has been inserted into the plasmid, it is cut with another restriction enzyme (Cla I) and ligated to the remainder of the adenovirus genome, which is provided as a pREP plasmid that has been linearized and is ready to be ligated.  The manual that accompanies the kit provides detailed instructions for designing hairpin sequences, inserting them into plasmid vectors, and for converting the plasmids into adenoviral vectors using the SpeAd™ method.

Kit includes:

pQuiet-U6 (20 ug)

pREP7, linearized (10 ug)

positive control plasmid to silence the expression of b-galactosidase (10 ug)

lambda phage packaging extract (5 x 50 ml)

phage dilution buffer (3 ml)

ready-to-use bacterial host (E coli DK-1 cells, 1 ml)

sequencing primers (2, 100 ng each)

SpeAd™ Manual