Nucleic acid handling

2.3.1 Small scale isolation of plasmid DNA

Small scale preparation of plasm id DNA w as perform ed according to the

2.3.2 Large scale isolation of plasmid DNA

Large scale isolation of p lasm id DNA w as p erform ed u sin g either the

W izard^^ Plus M axipreps D N A Purification System from Prom ega or the

alkaline lysis m ethod (Sambrook et a l, 1989).

2.3.3 Preparation of phage DN A

23.3.1 Preparation ofLE392 cells

A single colony of LE392 cells w as cultured overnight on a gyratory shaker

(set to 250rpm) in 50ml LB, su p p lem en ted w ith 0.2% m altose and lOmM MgSO^. The cells w ere pelleted by centrifugation at SOOOrpm for 10 min. at 4“C in a Sorvall GS-3 rotor. The su p ern atan t w as discarded and the cells resuspended in 15ml of ice-cold lOmM MgSO^.

2.3.3.2 Liquid culture of bacteriophage

For liquid culture, a bacteria to phage ration of 1000:1 is required. The

bacterial OD6oo=l for approxim ately 8 x 10® cells/m l. A rough estim ate is that

a phage plaque contains 1 0® plaque forming units (pfu).

A pproxim ately 2 x 10^ pfu w ere ad d ed to 200pi lOmM MgSO^ containing

approxim ately 2 x 10® bacteria, m ixing gently w ith the pipette tip. The

m ixture was incubated at 37°C w ith gentle shaking for 20 min. This w as then added to 500ml of LB (0.2% m altose, lOmM MgSO^) and cultured overnight at 37°C w ith shaking (250rpm).

2.3.3.3 Isolation of phage DNA

After checking for lysis, 10ml of chloroform w as added to the liquid culture. 50jLtl each of DNAsel and RNAseA (lO m g/m l) and 29g of NaCl w ere added and the m ixture incubated w ith vigorous shaking (>300rpm) at 37°C for 30 min. The contents w ere allow ed to settle before p o u rin g into tw o 500ml N algene centrifuge bottles leaving the chloroform behind. The bottles w ere centrifuged at 8000rpm in a Sorvall GS-3 rotor for 10 m in. at 4°C. The supernatant w as transferred into fresh bottles and 25g of solid PEG8000 w as dissolved into each. The m ixture w as then chilled to 4°C for at least one hour. This w as centrifuged at 8000rpm for 20 min. to recover the phage. The supernatant w as discarded, the pellet resuspended in 3ml of SM buffer and

transferred to a 30ml Corex tube. 1.5/d of DNAse and 30/d of RNAse w ere added and the m ixture incubated at 37°C for 30 min. 150/xl of 10% SDS, 120/xl of 0.5M EDTA (pH 8.0) and 30/xl of lO m g/m l proteinase K w ere added and

incubated at 6 8°C for 30 min. This was extracted, w ith equal volum es, first of

phenol, then 1 : 1 ph en o l/ch lo ro fo rm , and finally chloroform , before adding

1.5ml of 5M N H4OAC, 6ml ethanol and chilling on ice for 15 min. The tube

w as centrifuged at 9250rpm in a Sorvall SS34 rotor at 4 “C for 20 min., the

su p ern atan t discarded, the pellet air-dried and redissolved in 1.6ml H2O.

400/xl of 4M NaCl an d 2ml of 13% PEG8000 w ere ad d ed an d the m ixture chilled on ice for 1 hour to overnight. Following centrifugation at 9250rpm for 20 min. at 4°C, the su p ern atan t w as discarded, the pellet rinsed w ith 70% ethanol, air-dried and resuspended in 200-500/d dHzO.

2.3.4 Extraction and p recipitation of DNA and RNA

Extraction of DNA or RNA w ith phen o l/ch lo ro fo rm w as routinely used to rem ove contam inating proteins. A n equal volum e of either Tris.Cl (pH7.5)

equilibrated (for DNA) or DEPC H2O sa tu rated (for RNA) phenol and

chloroform (1:1) was added, m ixed vigorously and then the phases separated

by centrifugation at 12000g for 2 min. The u p p er (aqueous) phase was then rem oved and the DNA or RNA recovered by precipitation w ith ethanol. To one volum e of DNA, Vio volum e of 3M NaOAc (pH 4.9) and 2.5 volum es of ethanol w ere added. Following incubation either on dry ice for 15 min. or at -

20“C from V2 hour to overnight, the sam ple w as centrifuged at 12000g for 20

min. at 4°C. The nucleic acid pellet w as then w ashed w ith 70% ethanol and air-dried prior to resuspension.

2.3.5 Q uantitation of nucleic acid concentrations

Rough estim ates of nucleic acid concentration w ere m ade by com parison of sam ple m aterial w ith a sta n d a rd on an ag aro se gel. M ore accurate d e te rm in a tio n s w ere m ad e b y sp e ctro p h o to m e tric scan n in g at the

w av elen g th of 260nm. A n A2 6 0 value of 1 rep re se n ts an ap proxim ate

concentration of 50/ig/m l for double-stranded DNA, 40/ig/m l for single­ stranded RNA and 33/tg/m l for oligonucleotides.

2.3.6 Restriction enzyme digestion of DNA

Restriction digests w ere carried out according to the specifications of the m an u fa ctu rer of the p a rtic u la r restric tio n en d o n u clease (B oehringer M annheim or N ew England Biolabs). Sample DNA w as d ilu ted in sterile distilled w ater (SDW) and restriction endonuclease w as alw ays ad d ed in at

least a two-fold excess of the lU //zg recom m ended by Sambrook et al. (1989).

2.3.7 Electrophoretic separation of DNA

Following restriction endonuclease digestion, DNA sam ples w ere separated using agarose gel electrophoresis and TAE buffer (40mM Tris.acetate (pH 7.0), Im M EDTA). The concentration of agarose used depended on the size of

the fragm ents that w ere to be separated, b u t generally 1% gels w ere used

unless indicated otherw ise. DN A sam ples w ere loaded w ith one tenth volum e of 0.1% O range G and 50% glycerol into the wells. E thidium brom ide-stained DNA bands w ere visually detected w ith ultraviolet light and subsequently photographed. In order to purify DNA fragm ents from agarose gels, the QIAEXII Gel Extraction Kit (500) from QIAGEN w as used according to the m anufacturer's instructions. An alternative m ethod em ployed was to pierce a small hole in the bottom of a 0.5ml eppendorf tube, place first a small quantity of glass wool and then the excised gel fragm ent inside and to place the entire construct in a 1.5ml ep p en d o rf tube. This w as then sp u n at 6000rpm for 10 min. to elute the buffer containing the DNA from the gel fragm ent. The DNA was precipitated and resu sp en d ed in an appropriate volum e of sterile distilled water.

2.3.8 Blunt-ending, phosphatasing and ligation of DN A fragments

If fragm ents w ere to be ligated w hich had been digested w ith different restriction enzym es, the single-stranded overhangs w ere elim inated using one of two m ethods. To end-fill the overhang, the Klenow fragm ent of DNA

polymerase w as used as follows: O.IU of Klenow p er fil in a solution of lOmM

Tris (pH 7.4), 5mM MgClz and Im M of each of the four dNTPs, w hich was incubated at 37°C for 15 min. The DNA was then extracted an d precipitated.

To rem ove the overhang, m im g bean nuclease was used to digest the single­ stranded DNA. lOmM ZnClz w as ad d ed to the restriction digest to a final concentration of 1.5mM and m u n g bean nuclease to a final concentration of 0.1UjLtT\ Sufficient sterile distilled w ater w as added to keep the concentration of glycerol in the reaction to 10% or less. The reaction w as incubated at 30°C for 30 m inutes, and then gel-purified. Blunt-ended vector molecules (or those w hich had only been cut w ith a single restriction endonuclease) w ere treated w ith calf intestinal alkaline p h o sp h a ta se from B oehringer M annheim according to the m anufacturer's instructions to inhibit intram olecular ligation of vector w ithout insert. Vector an d insert fragments w ere mixed in a m olar

ratio of 1:3 and ligations w ere either carried out at 16“C overnight in 1 0/xl

reactions using T4 DN A ligase (100 ligation units for cohesive reactions) containing one tenth volum e of lOx ligase buffer (200mM Tris.Cl (pH7.6), lOOmM MgClz, lOOmM dithiothreitol, Im M ATP) or using Ready-To-Go™ T4 DNA Ligase from Pharm acia according to the m anufacturer's instructions.

2.3.9 D ouble stranded D N A sequencing

D ouble stran d ed p lasm id D N A w as sequenced (Sanger dideoxy chain term ination m ethod) u sing a Pharm acia ^^Sequencing kit according to the m an u factu rer's instructions. G lass sequencing plates w ere w ashed w ith detergent, rinsed well w ith tap w ater and finally w ith dHzO. They were then rinsed w ith 70% ethanol and left to air-dry before a final rub w ith chloroform. The surface of one plate w as treated w ith dim ethyldichlorosilane solution (BDH #33164) to prevent the gel sticking tightly to both plates. Plastic spacers (0.4mm) positioned along the sides separated the tw o plates w hich w ere clam ped together using bulldog clips and the plates w ere placed horizontally over a suitable object. The polyacrylam ide gel was prepared by mixing 100ml of 5% a cry lam id e/u rea solution (SEQUAGEL™ Sequencing System from N ational Diagnostics) w ith 800/^1 of 10% am m onium p ersu lp h ate solution and 40/xl of TEMED. The m ixture w as immediately draw n into a 50ml syringe and slowly injected betw een the horizontal glass plates, allow ing capillary action tw o draw the so lu tio n th ro u g h , assisted by tiltin g the plates as necessary. The flat sid e of a sh a rk 's tooth com b w as th en in serted

approxim ately 0.5cm into the solution and the gel allow ed to polym erise overnight. Electrophoresis w as carried out at a constant voltage of 25kV. The gel w as then fixed w ith 10% m e th a n o l/10% acetic acid for 30 min. before transferring onto a sheet of W hatm an 3MM p ap er an d dry in g u n d er a vacuum , using a Bio-Rad slab gel drier, for 1-2 hours. The labelled DNA fragm ents w ere visualised by autoradiography using K odak X-OMAT AR film.

2.3.10 Southern blotting

DNA to be blotted w as separated by agarose gel electrophoresis. The gel was photographed alongside a ruler to allow the calculation of fragm ent sizes from the autoradiograph. The gel was soaked twice for 30 min. in denaturing solution (0.5M N aO H , 1.5M NaCl) an d once, for a t least 10 min. in neutralising solution (0.5M Trizma base, 1.5M NaCl, p H 7.5). Blotting onto H ybond-N nylon m em branes (A m ersham ) w as p erfo rm ed by capillary transfer. A pre-w et wick of W hatm an 3MM paper (three layers) w as laid on a raised platform over a reservoir of 20xSSC (3M NaCl, 0.3M Tri-sodium citrate p H 7.0). The m em brane, soaked w ith 20xSSC, w as laid on top of this, followed by three sheets of 3MM paper (also saturated w ith 20xSSC) and then a stack of paper towel. This w as then w eighted dow n to aid the transfer. Following overnight transfer of DNA onto the filter, DNA w as crosslinked to the filter using a UV Stratalinker^^ 1800 (Stratagene) ultraviolet crosslinker

(energy setting of 1 2 0 0).