Transformation

Introduction

Transformation is a technique to introduce DNA into bacterial cells. There are many variations on a common theme, but the key points are listed below. Check details with supplier of competent bacteria and note that variations in timings and volumes will vary with application and bacterial strain.

There are four stages:

• Mix DNA/bacteria and incubate on ice Ð do not use an excessive amount of DNA, both in terms of concentration and actual volume (less than 1 μg and less than 10 μl). Note,protein (e.g. Ligase) will reduce transformation efficiency, but it is not always necessary to remove prior to transformation.
• Heat shock - necessary for DNA uptake. Time heat shock carefully - excessive heat shock will kill the bacteria and the transformation will fail.
• Recovery - prior to selecting for transformed bacteria with antibiotics, it is necessary to allow them to recover in rich medium (e.g. LB, SOC or 2YT) for 30-60 mins at 37 ûC.
• Selection - essential to isolate (as single colonies) the bacteria which have taken up DNA.This is usually performed on solid medium (LB-agar) in the presence of antibiotics. Cells are incubated at 37 ûC overnight.

Competent bacteria are extremely fragile - always thaw slowly on ice and do not hold the base of the eppendorf tube. The compency of the bacteria is also important - "sub-cloning efficiency" means about 106 colonies are produced per μg of (purified) DNA. "Library efficiency" can mean in excess of 109 colonies produced per μg of (purified) DNA. For sub- cloning and mutagenesis is normally sufficient, although "Library efficiency" bacteria may be useful if problems arise.

Bacterial Transformation

1. Add 1-10 μl of the DNA (Experimental reaction or positive/negative control) to a vial (20-200 μl) of competent E. coli cells and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 30 min.

3. Heat shock the cells for 30 sec at 42OC without shaking (time varies by strain).

4. Immediately transfer the tubes to ice and incubate for 2 min.

5. Add 50-500 μl nutrient broth (room temperature).

6. Cap the tube tightly and shake the tubes at 37ûC for 30 min. Place on ice.

7. Spread 50-500 μl from each transformation on a L-broth agar plates containing antibiotics at the appropriate concentration. Incubate plates for 5-10 mins at room temperature, then invert the plates and incubate overnight at 37OC.

Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satelite colonies will form. Plates/colonies can be stored for a few days at 4 OC if not to be used immediately.

Calculate transformation efficiency for the 1X and 10X DNA concentrations using the formula below.