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Protocols

Cloning Stem Loops into LentiLox 3.7

Cloning:

Oligos are resuspended in water at 60pmol/ul.

Annealing oligos:

1 ul Sense oligo

1 ul Antisense oligo

48 ul Annealing Buffer

Annealing Buffer:

100mM K-acetate

30mM HEPES-KOH pH 7.4

2mM Mg-acetate

Incubate at

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

Estimate concentration

Ligation

Ligate linearized product and annealed oligos at equimolar concentration. I typically use 60fmol of each component in a final concentration of 10uL.

Transformation:

I strongly recommend the use of an endA^- strain of E. coli. We have had success with STBL-2 cells.

Testing clones:

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:

5'-cagtgcaggggaaagaatagtagac-3'.

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.)