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 with 50 uL EB, mix: 43 uL purified PCR product 5 uL 10 x restriction enzyme buffer 1 uL of each restriction enzyme.

Alternatively, add directly to the tube with purified PCR product (slightly less than 50 uL), add: 5.5 uL 10x restriction enzyme buffer 1 uL of each restriction enzyme. (Suggestion only. Check if this method is suitable for routine use.)

NanoDrop:

To achieve more reliable results, follow the below procedure:

Initialize instrument Blank with water Measure concentration of DNA in water (should be close to zero) Measure concentration of DNA in buffer. Note if measurement on buffer gives a reading (wrongly) indicating DNA presence when using water as blank. Blank against buffer. Measure DNA concentration in buffer (should be zero).

Measure first sample. Wipe pedestal clean. Perform measurement using buffer as sample. Repeat until reading is close to zero. Measure next sample.

Blank