Molecular Biology Methods

2.2.1 Transformation of £.co/i

Preparation of Competent Cells

There are different ways o f introducing plasmid DNA into bacterial cells. These tend to vary in their

efficiencies and convenience. The method described below was easy and quick with moderate

transformation efficiencies (~ 10^transformants/|lg o f DNA).

This method relies on the dismption o f the bacterial cell wall by high concentrations o f calcium ions (Cohens

et a l, 1972). Cells should be prepared on the day before they are to be used as they will retain their competence for 24 hours. Cells can be resuspended in 15% glycerol at the final step and frozen in liquid

nitrogen. If kept at -20°C, cells in this form they will retain their competence for several months, but this

may be lower than for non-ffozen cells.

An aliquot ( 1 ml) o f an overnight culture o f the desired E. coli strain was used to inoculate 50ml o f sterile LB broth.

This fresh culture was incubated, with shaking, at 37°C to mid log phase (A^oo 0.3, approximately 2-3

hours) and harvested by centrifugation using a Sigma 6K10 centrifuge at 2400g for 25 minutes at 4°C and

then the supernatant was discarded.

The pellet was resuspended in a fifth o f the culture volume o f ice-cold 50 mM CaClj (25 ml) and stored

on ice for 30 minutes with frequent, gentle resuspension.

The cells were harvested again and resuspended in 2 ml o f ice-cold 50 mM CaCl2 ready for use.

Transformation of Competent E.co/f

Typically, between 2 and 200 ng o f plasmid DNA were added to 100 jll aliquots o f competent

cells in a sterile polypropylene tube and mixed gently. After incubation on ice for 45 minutes, the cells were

heat shocked by placing in a 42°C water bath for 90 seconds and returned on ice for further 5 minutes. The

tube was then placed at room temperature and 400 |il Soc (2% Bacto tryptone, 0.5% Bacto yeast extract,

10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO^, 20 mM glucose) was added to the tube and

mixed. The tube was then incubated with shaking for 45 minutes at 37°C before 1 OOjil was plated onto

the LB agar plate containing ampicillin. The plates were left to dry on the bench for 10-20 minutes before

being inverted and incubated overnight at 37°C. Controls for competence and sterility were always

included.

2.2.2 Ligation of DNA

Ligation reactions were carried out using an approximate molar ratio o f insert to vector o f 3:1. “Sticky end”

ligations were carried out in microfuge tubes in 10 |il with 1 unit o f ligase for every 100 ng DNA, 10 x

ligation buffer and 10 mM ATP or 100 mM ATP for “sticky end” ligation and the reaction was made up

with water. 1 pi o f T4 DNA ligase ( 1 U/pl) was added and mixed in. “Blunt end” ligations were incubated

for 16 hours at 16°C, while “sticky end” ligations were incubated for 2 hours at room temperature.

2.2.3 Restriction digestion of DNA.

Restriction enzymes were used according to manufacturer’s instmctions and inactivated after incubation by

treatment at 65°C.

The restriction reactions were carried out in microfuge tubes which, in general, contained 1 pg o f DNA,

1 pi o f restriction enzyme, 1 pi o f 1 Ox buffer and sterile HjO to give a final volume o f 10 pi. The reactions

were incubated at the required temperature fori -3 hours, inactivated and electrophoresed on an agarose

gel.

2.2.4 Ethanol precipitation of DNA Reagents:

TE buffer 1 OmM Tris/HCl (pH 8.0) ImM Na2EDTA (pH 8.0)

Ethanol precipitation was used to concentrate DNA solutions. Ethanol (250 |il) and 10 |ll o f 3 M sodium

acetate (pH 6.0) were added to the DNA solution which was made up to 100 |il with sterile H2O and

incubated at -20°C for 30 minutes. The mixture was centrifuged for 1 Ominutes in a microfuge and the

supernatant was removed. The pellet was washed with 300 |Il o f 70% ethanol, centrifuged for 5 minutes

and the supernatant was removed immediately. Care was taken as the pellet is less tightly packed at this

stage. After drying the pellet in air it was resuspended in an appropriate volume o f sterile TE buffer or sterile

H2O and stored at -20°C until required.

2.2.5 Small scale plasmid purification (Miniprep)

The method used for the small scale isolation and purification o f DNA was Wizard ™ Minipreps DNA

purification system from Promega.

DNA was prepared from 10 ml o f overnight cultures containing the desired plasmid. The kit was used

according to manufacturer’s instructions. DNA, purified from this method, was used in experiments

changing the reading frame o f pGEMEX-1 G48V “4833”.

2.2.6 Large scale plasmid DNA purification (Maxiprep)

This method was used to prepare stocks o f plasmid DNA for subsequent cloning. The method used for the

large scale isolation and purification o f DNA was Wizard™ Maxipreps DNA purification system fi*om

Promega. The kit was used according to manufacturer’s instructions. Since the E’.co//used, TGI strain

(described in table 2.1) contains Endonuclease A. which degrades DNA, further purification using

phenol;chloroform (described in 3.3.1.1) to purify the DNA was carried out.

2.2.7 Estimation of DNA concentration

The concentration o f a DNA solution was calculated by measuring its absorption at 260 nm and using the

equation.

Concentration (|Ig/ml) = Optical density (O.D) x dilution factor x 50

The DNA, for which the concentration was to be found, was firstly diluted, for example, by mixing 5 |ll o f

DNA solution with 495 |ll o f sterile HjO (i.ea 1:100 dilution) using matched quartz semi-micro cuvettes

( 1 cm pathlength) the absorbance was measured in a spectrophotometer (Cecil 2000) and this reading

allowed the calculation o f the concentration o f DNA using the above equation.

The absorbance was also measured at 280 nm, as the ratio between the two readings gives an estimate of

the purity o f the DNA. Pure DNA has an OD26o/OD2gobetween 1.8 and 2.0 for less pure DNA the ratio

is lower.

When the concentration o f DNA was too low to detect by this method, its’ concentration was estimated

by running a sample against a standard sample o f known concentration on an agarose gel and comparing

their fluorescence intensities following staining in ethidium bromide (0.1 |Ig/ml).

2.2.8 Agarose Gel Electrophoresis of DNA Reagents:

SOxTAE 242 g/1 Tris/HCl

100 ml/10.5M Na2EDTA (pH 8.0)

57.1 ml/1 Glacial Acetic Acid

6x loading solution 0.25% w/v bromophenol blue

30% v/v glycerol

Horizontal agarose gels were used for the routine identification, sizing and quantification o f DNA. Gels

containing 0.8% agarose (w/v) were used for the analysis o f DNA.

The agarose was dissolved in 1 x TAB by boiling in a microwave oven. Once the solution had cooled to

~ 45°C, ethidium bromide ( 10 mg/ml) was added to a final concentration o f 0.1 |Ig/ml and poured into the

Mini Sub DNA cell (Biorad) horizontal gel mould.

An 8 well comb was then inserted and the gel allowed to set. The gel was then placed in the gel tank and

TAB buffer added until the gel was submerged.

Samples were prepared by adding 0.2 volumes o f loading buffer and between 2 and 10 |Il o f samples were

loaded per well. Sample o f the 1 Kb ladder (Gibco-BRL) were loaded as molecular weight markers. Gels

were run at 100 V until the dye front had migrated three quarters o f the way along the gel.

2.2.9 Visualisation and photography of DNA on Agarose Gels

The DNA was visualised by placing the gel on a UV transilluminator. The gel was photographed using a

Polaroid camera fitted with Kodak Wratted 23A filter and polaroid type 667 film.

The film was exposed for 30 seconds at f 11 and processed according to the manufacturer’s instruction.

2.2.10 Recovery o f DNA from Agarose gels ( Band Extraction”)

The desired band was visualised on the gel using a UV transilluminator and a clean scalpel blade used to

cut out a piece o f the gel containing the band. The DNA was recovered from the gel slice by the Wizard™

DNA clean-up system from Promega. The method was carried out according to the manufacturer’s

instruction.

2.3

Protein Chemistry Methods