TITLE: Cloning shRNA Oligos into pLKO.1
AUTHORS: Addgene  The Nonprofit Plasmid Repository

[DESCRIPTION]
This is the protocol accompanying the "pLKO.1 – TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: http://www.addgene.org/tools/protocols/plko/

[STEPS]
SECTION: Annealing Oligos
1. Resuspend oligos in ddH2O to a concentration of 20 μM.
SECTION: Annealing Oligos
2. Add 5ul Forward oligo 
5 1
SECTION: Annealing Oligos
3. Add 5ul Reverse oligo 
5 1
SECTION: Annealing Oligos
5. Add 35 μL ddH2O 
35 1
SECTION: Annealing Oligos
6. Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling water. 
4 min
SECTION: Annealing Oligos
7. Incubate the sample at 70°C for 10 minutes in a PCR machine. 
10 min
SECTION: Annealing Oligos
8. Slowly cool to room temperature over the period of several hours. 
180 min
 
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
9. Mix: 6 μg pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) 
6 4
SECTION: Digesting pLKO.1 TRC Cloning Vector
10. with 5 μL	10x NEB buffer 1 
5 1
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
11. with 1 μL	AgeI 
1 1
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
13. Incubate at 37°C for 2 hours. 
120 min
SECTION: Digesting pLKO.1 TRC Cloning Vector
14. Purify with Qiaquick gel extraction kit, eluting in 30 μL of ddH2O.
SECTION: Digesting pLKO.1 TRC Cloning Vector
15. Digest eluate with EcoRI by mixing: 30 μL pLKO.1 TRC-cloning vector digested with AgeI
SECTION: Digesting pLKO.1 TRC Cloning Vector
16. with 5 μL	10x NEB buffer for EcoRI 
5 1
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
17. with 1 μL	EcoRI 
1 1
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
18. and 14 μL	ddH2O 
14 1
SECTION: Digesting pLKO.1 TRC Cloning Vector
19. Incubate at 37°C for 2 hours. 
120 min
SECTION: Digesting pLKO.1 TRC Cloning Vector
20. Run digested DNA on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb.  
 
SECTION: Digesting pLKO.1 TRC Cloning Vector
21. Cut out the 7kb band and place in a sterile microcentrifuge tube.
SECTION: Digesting pLKO.1 TRC Cloning Vector
22. Purify the DNA using a Qiaquick gel extraction kit. Elute in 30 μL of ddH2O.
SECTION: Digesting pLKO.1 TRC Cloning Vector
23. Measure the DNA concentration.
SECTION: Ligating and Transforming into Bacteria
24. Use your ligation method of choice. For a standard T4 ligation, mix: 2 μL	annealed oligo from "Annealing Oligos" section above. 
2 1
SECTION: Ligating and Transforming into Bacteria
25. With 20 ng digested pLKO.1 TRC-cloning vector from the "Digesting pLKO.1 TRC Cloning Vector" section above. 
20 8
 
SECTION: Ligating and Transforming into Bacteria
26. With 2 μL	10x NEB T4 DNA ligase buffer 
2 1
 
SECTION: Ligating and Transforming into Bacteria
27. With 1 μL	NEB T4 DNA ligase 
1 1
 
SECTION: Ligating and Transforming into Bacteria
28. Bring up to 20ul with ddH2O
SECTION: Ligating and Transforming into Bacteria
29. Incubate at 16°C for 4-20 hours. 
240 min
SECTION: Ligating and Transforming into Bacteria
30. Transform 2 μL of ligation mix into 25 μL competent cells, following manufacturer’s protocol.

 

SECTION: Ligating and Transforming into Bacteria
31. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin (an ampicillin analog).
SECTION: Annealing Oligos
4. Add 5ul 10X NEB Buffer 2  
5 µL 

SECTION: Digesting pLKO.1 TRC Cloning Vector
12. Bring up to 50  μl with ddH2O