DNA Methods

2.2.1 Restriction enzyme digestion

Restriction digests were carried out in buffers recommended by the manufacturer.

Typically, 5pg of DNA was digested with 2p,l enzyme for at least one hour, in a total

volume of lOOp-1 (lOpl buffer, the rest dH20). Following digestion, the DNA was

precipitated for at least 30 minutes at -20°C, in 200pl 100% ethanol and lOp.1

NH4AC. After centrifuging, the pellet was resuspended in lOpl dH20 and a Ipl

sample was run on a 1% agarose gel.

2.2.2 Agarose gel electrophoresis of DNA

Normal agarose (SIGMA) was used for the preparation of gels. Restriction digests

were analysed on horizontal gels containing 1% (w/v) agarose in Ix Tris-Acetate-

EDTA (TAE) buffer, with 0.5pg/ml ethidium bromide (final concentration). Samples

were mixed 2:1 (v/v) with agarose gel loading buffer, loaded onto the gel, and

electorophoresed at 5 volts/cm in TAE buffer. The gel was stopped when a clear

band of nucleic acid could be visualised on a short wave transilluminator (254nm),

2.2.3 Preparation of competent cells

A single colony from an LB agar plate was used to innoculate 2.5ml LB broth and

incubated overnight at 37°C. The following day, the entire overnight culture was

used to innoculate 250ml LB broth containing 20mM MgSO^. The cells were grown

in a 1 litre flask until the A^oo reached 0.4- 0.6 (typically 1-2 hours). The cells were

harvested by centrifugation at 4500x g in a Beckman J25 for 5 minutes at 4°C. They

were then gently resuspended in 100ml ice-cold TFBl and incubated on ice for 5

minutes. The cells were again centrifuged at 4500x g for 5 minutes at 4°C, and

gently resuspended in 10ml ice-cold TFB2. Finally, the cells were incubated on ice

for 15-60 minutes. They were then split into 200pl aliquots, quick-frozen in a dry

ice/ isopropanol bath and stored at -70°C.

TFBl: 30mM KAc, lOmM CaClj, 50mM MnClz, lOOmM RbCl, 15% glycerol,

made up in dHjO.

TFB2: lOmM MOPS, 75mM CaCl2, lOmMRbCl, 15% glycerol, made up in CIH2O.

2.2.4 Transformation of competent cells with plasmid DNA

50pl competent cells and lOng plasmid DNA were added to chilled transformation

tubes. After swirling to mix, the tubes were stood on ice for 45 minutes. The cells

were heat-shocked at 37°C for 45 seconds, and then chilled on ice for 2 minutes.

0.5ml LB broth was added to each tube and they were incubated at 37°C for at least 1

hour. 50|jil was plated onto ampicillin selective LB agar plates and incubated

2.2.5 Large scale plasmid preparation

The procedure for plasmid maxi-preps outlined here is based upon the method

described in (Sambrook et a l, 1989).

A single colony from an ampicillin selective LB agar plate was used to innoculate

500ml LB broth (with 500pl ampicillin stock), and incubated overnight in a 3 litre

conical flask at 37°C. The following day, the culture was transferred to 500ml

nalgene bottles and centrifuged at 3000x g (4000 rpm) in a Beckman JLA 10,500

rotor for 15 minutes. The media was removed and the pellet resuspended in 20ml

chilled Solution I, with vortexing. 40ml fresh Solution II was then added to lyse the

cells, and the tubes gently inverted to mix. After leaving at room temperature for 5-

10 minutes, 20ml ice-cold Solution II was added and the tube softly shaken 3-4

times. A snowy precipitate of protein and chromosomal DNA was immediately

formed. After leaving on ice for 10 minutes, the tubes were again centrifuged at

3000x g for 15 minutes at 4°C. The supernatant was filtered through a tissue into a

fresh nalgene bottle, leaving a clear liquid. 0 .6 volumes of isopropanol was added to

precipitate the DNA and, after vortexing, this was left at room temperature for 10

minutes. The DNA pellet was recovered by centrifuging at 3000x g for 15 minutes

and discarding the supernatant. The pellet was washed with 70% ethanol and left at

room temperature to dry.

Solution I: 50mM glucose, 25mM Tris-HCl pHS.O, lOmMEDTA.

Solution II: 0.2N NaOH, 1% SDS.

Solution III: 60ml 5M potassium acetate, lL 5m l acetic acid, made up to 100ml

The DNA pellet was dissolved in 5.6ml TE (pH8.0), and transferred to a 50ml Falcon

tube wrapped in foil. To this was added 6g CsCl and 430|xl EthBr of a lOmg/ml

stock. The solution was loaded into a Beckman centrifuge tube, leaving no air, and

the top sealed. The tubes were very carefully balanced and centrifuged for 16 hours

at 50,000 rpm in a Beckman Ti 70.1 rotor. The following day, the band of DNA was

carefully removed using a syringe and transferred to a foil-wrapped Sterilin tube. An

equal volume of water-saturated butanol was added, and the mixture shaken and left

to separate. The bottom layer was removed with a pipette and put into a new Sterilin

tube, with another volume of butanol. This was repeated until neither layer was pink.

Finally, the lower layer was taken and to it was added 3 volumes of dHjO plus 8

volumes cold 100% ethanol. The mixture was vortexed and left on ice for 20

minutes. The pellet of DNA was recovered by centrifuging at 4800 rpm (3800x g) in

a SIGMA 3K10 benchtop centrifuge for 15 minutes at 4°C. The pellet was washed

with 70% ethanol, dried and resuspended in 400|l i1TE. The DNA was quantitated

using a spectrophotometer, by measuring the absorbance of an aliquot of the solution

at 260 and 280nm. Its quality was also checked by gel electrophoresis, after which it

was stored at a concentration of Ipg/pl, at -20°C. This method typically gave

approximately 1.5mg extremely clean DNA.

2.2.6 Synthesis of RNA probes

The reagents were mixed together at room temperature in the following order:

Sterile distilled water lOpl

5x transcription buffer 4pl

Nucleotide mix (lOmM each of GTP, ATP, CTP and 6.5mM UTP, 3.5mM

digoxygenin-UTP pH8.0) 2pl

Linearised plasmid (Ipg/jLil) 1 or 2pl

Placental ribonuclease inhibitor (lOOU/ml) 0.5pl

SP6, T3 or T7 RNA polymerase (lOU/ml) Ipl

The mixture was incubated at 37°C for 2 hours. After 1 hour, a further Ip l of

polymerase was added. RNA was precipitated by adding 100|il TE, lOpl 4M LiCl,

300pl 100% ethanol and Ipl tRNA. This solution was incubated at -20°C for at least

30 minutes, and then centrifuged for 15 minutes. The pellet was washed in 70%

ethanol, air dried and resuspended in lOpl TE. A 2p,l aliquot was taken and run on a

1% agarose gel, to check the size of the band and estimate the concentration of RNA.

Prehyb mix (see section 2.4.1) was added to the remaining 8pi of probe, in order to

obtain a concentration of Ipg/lOOpl. Probes were then stored in this manner at -

70°C.