|Lentiviral RNAi Protocols: Cloning
Identification of stem loop sequences:
Oligos are resuspended in water at 60 pmol/ml.
1 ml Sense oligo
Incubate at 95° 4min
Decrease temperature to 4° slowly (.1°C/min)
Incubate at 4° 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. Use 60fmol of each component in a final concentration of 10 mL.
Use of an endA- strain of E. coli is strongly recommended. 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. )
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