|Lentiviral RNAi Protocols - Cloning|
Oligos are resuspended in water at 60pmol/ul.
1 ul Sense oligo
1 ul Antisense oligo
48 ul Annealing Buffer
30mM HEPES-KOH pH 7.4
95C for 4min
70C for 10min
Decrease temperature to 4C slowly (.1C/min)
Incubate at 4C for 10 min
Digestion of pLentiLox 3.7
Digest 1-2ug with XhoI and HpaI
Treat with SAP or with CIP
Purify linearized fragment
Ligate linearized product and annealed oligos at equimolar concentration. I typically use 60fmol of each component in a final concentration of 10uL.
I strongly recommend the use of an endA- strain of E. coli. We have had success with STBL-2 cells.
We have had success testing for insertion of the stem-loop sequence with both colony pcr or by restriction digest. Insertion of insert causes a band shift of ~60bp in an XbaI/NotI fragment when compared to parental vector. This can be seen by 2% agarose gel electrophoresis. The following primer which corresponds to FLAP can be used to sequence into the U6 promoter and stem loop:
Preparation of DNA:
We recommend the use of Qiagen
Endo-Free Maxiprep Kits for all plasmids used
in transfection of 293.T cells (293FT cell line is available from Invitrogen,
Catalog #R700-07. )
|This webpage was created by Chris Dillon (modified by AED)|