This is a simple subcloning protocol which uses PCR primers to add new restriction sites to the ends of the cloned DNA segment. Since the vector is not dephosphorylated, it is important that the two different restriction enzyme sites are used at the ends. Unlike most similar protocols, no phenol-chloroform extraction steps are required.
1. Amplify template: perform 4 PCR reactions per template using Platinum Hifi Taq (Invitrogen):
- 5 ul 10x Hifi buffer
- 1 ul 10 mM dNTPs
- 2 ul 50 mM MgSO4
- 1 ul 10 uM forward primers
- 1 ul 10 uM reverse primers
- 0.5 ul Taq
- 37.5 ul H2O
- 2 ul 10 ng/ul template DNA
Program: 94 C 2 min, (94 C 30 sec, 45 C [5 C lower than melt temp] 30 sec, 68 C 90 sec)*30
The region of the PCR primers that binds the template should have a melting temperature of ~50 C (+/- a few degrees). The 5′ end of the primers should contain the new restriction site as well as additional bases to ensure efficient cleavage near the end of the PCR product (see NEB catalog).
2. Pool PCR reactions, clean using Sigma Genelute PCR clean-up kit (follow protocol except elute in 50 ul 10 mM TRIS, not TE)
3. Digest ~4 ug PCR products (20 ul of clean PCR product) and ~5 ug of destination vector using 40 U of each restriction enzyme in a total volume of 40 ul.
4. Run digest products on 1% agarose gel.
5. Cut out appropriate bands and purify DNA using Invitrogen gel extraction kit (elute using 10 mM TRIS, not TE)
6. Run diagnostic agarose gel with 2.5 ul of undiluted and 5x diluted extracted DNA. Quantify DNA based on band intensity.
7. Perform ligations using NEB quick ligation kit. Follow protocol (use 3-fold molar excess of insert). If desired, perform additional ligations with differnet amounts of insert DNA.
Perform a control ligation with the vector alone.
8. Transform competent E. coli by heat shock.