1. pSilencer™ 4.1-CMV puro siRNA Expression Vector Kit which includes:
   • Linearized pSilencer™ 4.1-CMV puro vector ready for ligation, 20ul @ 0.1ug/ul
   • Circular, negtive control pSilencer™ 4.1-CMV puro vector that expresses a hairpin siRNA with limited homology to any known sequeces in the human, mouse and rat genomes, 10ul @ 0.5ug/ul
   • GAPDH-specific, hairpin siRNA insert that can be used as a positive control for ligation, 10ul @ 80ng/ul
   • 1x DNA Annealing Solution to prepare annealed DNA olidonucleotides for ligation into the pSilencer™ 4.1-CMV puro vector, 1ml
2. 55 mer oligonucleotide pairs (See further reading and tools part for design and order)
   • Using any siRNA design program, search and choose three pairs of 21mer siRNA sequeces, convert to the 55mer sequences for the vector with Ambion design tool. and order 25nM desalted oligonucleotides.
   • Dissolve oligos with T₁₀8.0 and store at -80^oC. Dilute to 1ug/ul before use.
3. Ligation and transformation reagents:
   • T4 DNA ligase and ligase reaction buffer (-20^oC)
   • Competent E. coli cells (NovaBlue, -80^oC)
   • Ampicillin containing plates and liquid media (LB) (4^oC)
4. Plasmid purification reagents
   • Qiagen Plasmid Mini Kit for primary plasmid DNA extraction and iditification
   • Qiagen Plasmid Midi or Maxi Kit for the plasmid DNA used in transfection
5. Restriction analysis reagents and equipment
   • For initial clone verification, the restriction enzymes Bam H1 and Hind III are used.
   • agarose gel electrophoresis materials and apparatus
6. Sequence analysis
   • Putative clones containing pSilencer 4.1-CMV puro with shRNA template
   • Primers for insert sequencing: 5'-aggcgattaagttgggta-3' and 5'-cggtaggcgtgtacggtg-3'
   • Send the samples at asigned concentration and buffer to DNA Sequencing Facility
7. Mammalian cell transfection reagents
• FuGENE 6 Transfection Reagent or Lipofectamine 2000
8. Cell culture facility and supplies
   • Larmina flow hood, CO2 Incubator, Tissue culture dishes, flasks, serological pipette
9. Other
   • RNase-free tubes and racks, Centrifuge, ThermoMixer, Gilson Pipette and aerosol tips, Incubator (37^oC for bacteria culture), Gloves...
   • ice

1. Do a Puromycin titration (kill curve) on the cells to be transfected. at concentration of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3, 5, 7, 10, 15 and 20 ug/ml. For most cells used in the lab the puromycin will fall into the concentration between 0.5 and 5ug/ml.
2. Annealing:
   a. Assemble the 50 µl annealing mixture as follows:

b. Heat the mixture to 90°C for 3 min, then cool to 37°C and incubate
for 1 hr at 37°C.
c. The annealed hairpin siRNA template insert can either be ligated
into a pSilencer 4.1-CMV vector immediately or stored at –20°C.

3. Ligation
   a. Dilute 5 µl of the annealed hairpin siRNA template insert with 45 µl
   nuclease-free water for a final concentration of 8 ng/µl.
   b. Set up two 10 µl ligation reactions for each annealed oligo pairs; a plus-insert ligation, and the minus-insert negative control. To each tube, add the following reagents:
   

   Component	Plus-insert	Minus-insert
   diluted annealed siRNA insert	1 µl	-
   1X DNA Annealing Solution	-	1 µl
   Nuclease-free water	6µl	6µl
   10X T4 DNA Ligase Buffer	1 µl	1 µl
   pSilencer 4.1-CMV puro vector	1 µl	1 µl
   T4 DNA ligase (5 U/µl)	1 µl	1 µl

   3. Incubate at 15^oC on ThermoMixer overnight.
4. Transformation
• Transform 3-5ul each ligation product into competent cells with pSilencer vector alone as control (refer to ligation and transformation protocols).
5. Identify clones with the hairpin siRNA insert
• Pick clones, isolate plasmid DNA, and digest the plasmid with BamH I
  and Hind III to confirm the presence of the ~55 bp siRNA insert.
  To further confirm, send the plasmid DNA with the sequencing primers to the Sequencing Facility.
6. Make a Maxi Prep of each selected pSilencer 4.1-CMV plasmid for transfection
7. Transfect cells and select puromycin resistant colonies.
8. Grow the cells and the gene silencing can be tested by Western blot or qRT-PCR.
   

Further Reading and tools

• Ambion pSilencer 4.1-CMV puro Instruction Manual (pdf)
• Ambion RNAi resources /Design Tool for the pSilencer™ Vectors
  • The design tool actually is a converter from 21mer siRNA or DNA sequences to the sequences fitting the pSilencer vectors. Ambion probably is only the one who does'nt provide its siRNA sequences to the public among the companies providing RNAi products.
• NCBI
• IDT ddRNAi design
• Invitrogen
• Qiagen
• Promega