User:Jarle Pahr/Protocols

Links and notes on basic protocols:

Mixing of dNTPs.

A 10 mM dNTP mix should contain 10 mM dNTPs total (2.5 mM of each). To prepare 40 uL 10 mM dNTP, mix:

1 uL 100 mM dATP 1 uL 100 mM dGTP 1 uL 100 mM dCTP 1 uL 100 mM dTTP 36 uL H2O.

Ligation:

~100 ng equivalents backbone DNA. 3:1 - 5:1 molar ratio insert/backbone 2 uL T4 DNA ligase buffer 0.5 uL T4 DNA ligase H2O to 20 uL

Incubate at 16 C for 3 h or overnight.

Transformation of E. coli:

Transform using 1 uL plasmid DNA added to 0.1 mL supercompetent cells. For ligation mixtures, use 2-5 uL. Incubate on ice for 30 min. Heat shock at 42 C for 45 s. Incubate 2 min on ice, then add 200 SOC or LB medium. After heat-shock and addition of medium, incubate 30 min - 1 h for Kan-resistant cells. Spread 100 uL per plate (maximum 200 uL per plate).

Restriction digest:

General purpose restriction digest of purified PCR product:

After purification of PCR product and elution in 50 uL EB, mix: 43 uL purified PCR product 5 uL restriction enzyme buffer 1 uL of each restriction enzyme.