1. Search for the gene(s) of interest on The RNAi Consortium (TRC) portal (www.broadinstitute/
rnai/public/) and lect ~five non-overlapping targeting quences.
•In general, quences targeting the CDS are preferred becau they can be rapidly screened by transient co-transfection with an expression plasmid for the gene to be knocked down •Sequences that are 100% human specific are preferred so that future addback experiments can be performed with the mou or rat homolog
•Custom targeting quences for special applications can be designed according to the TRC design rules: www.broadinstitute/science/projects/rnai-consortium/trc-shrna-design-process
2. Order the primers (minimum synthesis, standard desalting) listed under “Oligo design for arrayed
cloning” after replacing the XhoI site in the loop (CTCGAG) with a PstI site (CTGCAG). Dissolve primers to a concentration of 50 µM in PCR-grade H2O
•Unlike XhoI, PstI restriction sites are abnt in pLKO.1, allowing ligated hairpins to be screened rapidly by restriction digest
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3. Anneal the shRNA primers by tting up the following reaction in a PCR tube (one annealing reaction
per shRNA duplex):
• 2.5 µl top-strand primer
• 2.5 µl bottom-strand primer
• 5 µl 10× annealing buffer
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•40 µl ddH2O
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50 µl total volume
4. Heat the annealing reaction to 95o C for 5 min in a PCR thermocycler. Unplug the thermocycler and
allow the primers to anneal by cooling to room temperature overnight.
•The shRNA oligos are designed to contain restriction enzyme-like cleavage ends after annealing;
therefore, they do not need to be digested
5. Phosphorylate the annealed shRNA primers by tting up the following reaction in a PCR tube:
• 1 µl annealed primers
• 1 µl 10 mM ATP
• 1 µl 10× PNK reaction buffer (supplied with NEB #M0201)
• 1 µl T4 polynucleotide kina (NEB #M0201)
• 6 µl ddH2O
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10 µl total volume
6. Incubate the phosphorylation reaction at 37o C for 30 min, then heat inactivate at 70o C for 10 min.
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7. In parallel with the oligo annealing and phosphorylation, t up three 50 µl restriction digests, each
containing 2 µg pLKO.1, 5 µl 10× EcoRI buffer, 2.5 µl EcoRI, 2.5 µl AgeI, and 0.5 µl of 100× BSA.
•We have DNA stocks of pLKO.1 puro, pLKO.1 neo, and pLKO.1 hygro, which can be ud depending on the combination of perturbations desired
8. Digest pLKO.1 at 37o C for 3 hr and purify the ~7 kb digested fragment on a 1% agaro gel
•The pLKO.1 ries releas a 1.9 kb “stuffer” quence after EcoRI-AgeI digest, which readily parates the double-cut vector from any single-cut vector
9. Exci the digested vector, purify, and ethanol precipitate the three digests into 5 µl EB as described in
Janes_PCRcloning.pdf.
10. Set up a 10 µl ligation reaction containing 7.5 µl phosphorylated-annealed shRNA oligo, 100–250 ng
digested pLKO.1, 1 µl 10× T4 ligation buffer, and 0.5 µl T4 DNA liga (New England Biolabs
#M0202S).
•Be sure that the ligation buffer has thawed completely (no visible precipitates) and is kept at room temperature before u
未来黑科技•Note this reaction volume is half that recommended by the manufacturer
•Be sure to include a control ligation that includes everything but the shRNA oligo
加入英文11. Mix by pipetting gently, incubate at room temperature for 1 hr, and then ethanol precipitate and
transform into electrocompetent bacteria as described in Janes_PCRcloning.pdf.
12. Perform a PstI-BamHI diagnostic digest on ~two ampicillin-resistant colonies per shRNA oligo. Positive
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clones will yield two products at ~6.3 kb and ~700 bp.
13. Screen hairpins by transient transfection in 293T cells (if possible) or by transduction with lentivirus怎么打开运行窗口
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as described in Janes_Virusprep.pdf.
Buffer recipes
•10× annealing buffer Store at room temperature 100 mM Tris-HCl (pH 7.5)
1 M NaCl
10 mM EDTA
