Molecular Cloning

2.1.1.1 Restriction digests

Plasm id D N A (2-3|ig) was normally digested in a volume o f 3 0|l i1 w ith N ew E n g la n d B io la b s e n z y m e s (u sin g the

m anufacturer's buffers) for l- 2 h at 25-37°C. F rag m en ts were separated by agarose gel electrophoresis.

2.1.1.2 Klenow treatment

Klenow polymerase was used to fill protruding D N A ends after digestions. The DNA was incubated with Im M dNTPs and 2 units D N A Polym erase I (K lenow fragm ent, New E ngland Biolabs), in high salt buffer (lOOmM NaCl, 50m M T ris-H C l (pH7.5), lOmM M gCl2, ImM DTT) for lOmin at 16°C followed by lOmin at 37°C. As Klenow Polym erase is heat stable, the DNA was purified using p henol/chloroform extraction.

2.1.1.3 Phosphatase treatm ent

To avoid religation of linearised vectors, DN A was treated with 1 unit of Calf Intestinal Phosphatase (New England Biolabs) in p h o sp h a tase b u ffer (lOx buffer: 0.5M T ris-H C l (pH 9.0), lOmM M g C l2, Im M ZnCl2, lOmM Sperm idine) for 15-30min at 37®C to remove 5' phosphates. The enzym e was rem oved using p h e n o l/c h lo ro fo rm ex tra ctio n .

2.1.1.4 Kinase treatment

O lig o n u cleo tid es w ere 5 '-p h o sp h o ry la te d b efo re ligation. The D N A was treated with 5 units T4 p o ly n u cleo tid e kinase (Boehringer Mannheim) in 5mM Tris-HCl (pH7.4), Im M M gC l2, 5mM DTT and Im M ATP. After 30 minutes at 37°C, the kinase was inactivated at 7QoC for 15min.

2.1.1.5 Agarose gel electrophoresis

For fragments between 500 and 5000 basepairs (bp), a 1% agarose gel was used. F rag m en ts o f less than 5 00bp w ere separated on a 1.5-2% gel. For an aly tical p u rp o se s n o rm al agarose (Gibco BRL) was boiled in 50ml IxT B E (lOxTBE: 162g Trizm a base, 27.5g borate, 9.5g E D T A in IL water), ethidium bromide (0.5mg/ml) was added and the solution left to solidify in a gel plate. If the DNA fragments were to be used directly for lig a tio n , lo w m e ltin g p o in t a g a r o s e (se a p l a q u e , EM C B io P ro d u c ts , C a t-N °. 50102) was used. The D N A sam ple was loaded in 0.1 volume of lOx loading buffer (40% glycerol, 0.4% brom ophenol blue) and electrophoresis was carried out at 120-

130mA for about Ih.

2.1.1.6 Ligation of DNA fragments

Ligations were usually carried out in low m elting point agarose, a fter m eltin g and a d d itio n o f lOx lig a tio n b u ffe r (200mM Tris-HCl (pH7.4), lOOmM M g C h , lOOmM DTT, lOOmM ATP). The m olar ratio of vector to insert was betw een 1:5 and 1:10 (ty p ically 10-20ng vecto r : Zoong insert). L ig a tio n s w ere p e rfo rm ed for 6-15h at 16®C for sticky ends and at room tem perature for blunt ends. The ligation m ixes were "cleaned up" by dilution in 400^1 of TE (pH7.5) (lOmM Tris-HCl (pH7.5), 0.4mM EDTA) followed by extraction first with an equal volume o f P h e n o l/C h lo ro fo rm /Is o a m y l A lc o h o l (2 4 /2 4 /1 ), then w ith c h lo ro fo rm /Is o a m y l a lc o h o l (2 4 /1 ), T he D N A w as e th a n o l precipitated and resuspended in sterile double distilled water.

2.1.1.7 Preparation of competent cells

Electrocom petent stocks of D H 5 a and XL-1 blue cells were made using the following protocol. Cells were streaked from a glycerol stock onto an LB-Agar (L-broth + 1.5% agar) plate and incubated overnight at 37°C. One colony was picked from the plate and grown overnight in 10ml o f LB in a 37°C shaker. The overnight culture was diluted in the m orning into I L of LB, and grown at 37°C until an OD^OO of 0.6 was reached. The cells were transferred to four 250 ml sterile Corning centrifuge tubes, left

to cool on ice for 5m in, and p e lle te d at 3 0 0 0 rp m in a refrigerated Beckm an JA -20 centrifuge at 4°C for 30min. The cell pellet was resuspended in IL o f ice-cold sterile water. The procedure was repeated first with 500m l o f water, then with 2 0 ml 10% g ly ce ro l in w a ter and fin a lly the c ells w ere resupended in 2.5ml of ice-cold 10% glycerol. SOfxl aliquots were snap-frozen in liquid nitrogen, and stored at -80°C for up to 6 months. Transform ation efficiencies o f l O ^ c o l o n i e s / p g of plasmid DNA were usually achieved.

2.1.1.8 T ransformation

1 - l O p l of DNA to be transformed were added, on ice, to 4 0 p l of electrocompetent cells in a microfuge tube, m ixed gently and transferred to a 0.2cm electroporation cuvette (Biorad). The cells were electroporated on a Biorad instrum ent at 2.5Kv, 200 Omhs and 25|iF. The cells were transferred to a 5ml universal tube with 1ml of LB and incubated in a 37°C shaker for Ih. Cells were p e lle te d at 13000rpm , re s u s p e n d e d in 1 0 0 |il LB and plated on an LB-Agar plate containing lOOpg/ml Am picillin. The cells w ere in c u b a te d o v e rn ig h t and m in ip re p s m ad e from individual colonies to analyse DNA incorporation.