HOW TO CLONE IN THE MORRISEY LAB

The following is a short manual on how we clone DNA into plasmids. These techniques are not to be taken for granted and if one follows the instructions closely, they should be successful!

Cloning into plasmids

1. Digest 2mgs of plasmid DNA (1 hour) in 20ml volume. Dephosphorylate 5’ overhang ends as follows:

• heat inactivate restriction enzymes at 65⁰C for 15 min.
• add 4ml of 10X calf intestinal phosphotase buffer, 1.5 ml of calf intestinal phosphotase enzyme and 15ml of dH₂0. Incubate at 37⁰C for 1 hour.
• If DNA ends are blunt or have a 3’ overhang, see dephosphorylation of blunt ends protocol.

1. Digest either plasmid DNA or PCR products (digest for 2 hours) to generate insert for cloning-generally want 500-1000 ngs of insert DNA.

2. Run dephosphorylated plasmid and insert DNA on a low melting temperature agarose gel and cut out appropriate bands. Melt gel slices at 65⁰C for 10 minutes and extract 2 times with buffer saturated phenol and once with CHCl₃:isoamyl alcohol and then precipitate with 3 volumes EtOH, 1/10^th volume NaAcetate and 1ml glycogen.

3. Resuspend plasmid DNA in 20ml of TE and insert DNA in 6ml of dH₂0. Set up ligation as follows:

• control ligation: 6ml of dH20, 2 ml of 5X T4 Ligation buffer, 1ml of digested and dephosphorylated plasmid DNA (approx. 100ngs), and 1.5ml of T4 ligase enzyme.
• Insert ligation: 6ml of insert DNA (see above), 2 ml of 5X T4 Ligation buffer, 1ml of digested and dephosphorylated plasmid DNA (approx. 100ngs) , and 1.5ml of T4 ligase enzyme.
• for 5’ or 3’ overhand ends, ligate for 1 hour at room temperature
• for blunt end ligation, ligate overnight at 15⁰C.

5) Electroporate E.coli and plate on LB/amp or LB/carb plates (or different antibiotic if necessary).