El teorema fundamental del álgebra en Descartes

Bacteriophage T4 DNA polymerase (Roche) or the Klenow fragment of E. coli DNA polymerase I (Roche) were used to blunten appropriately digested DNA inserts or vectors as described to Sambrook et al. ( 1 989). The recessed 3' termini were end-filled by the DNA polymerase activity of the Klenow fragment of E. coli DNA polymerase I. Both Bacteriophage T4 DNA polymerase and the Klenow fragment of E. coli DNA polymerase I have 3'-5' exonuclease activity that was used to remove protruding 3' termini.

Chapter 2, Methods and Materials ₅₀

2.2.6 Ligation reactions

Ligation reactions were carried out using 1 unit T4 DNA ligase (Roche or Promega) per 100 ng vector DNA, and the appropriate volume of 10 x or 2 x ligation buffer supplied by the manufacturer. The standard T4 DNA ligase reaction using 10 x buffer was incubated at 4°C for 16 h. Similar reactions using 2 x rapid ligation buffer were complete after 5 min incubation at ambient room temperature. Molar ratios of 1 : 1 . 1 : 3 and 3 : 1 of vector:insert DNA were routinely used. Some blunt ligations required higher insert concentrations to drive ligation reactions so molar ratios of 1 :5 and 1 : 10 of vector: insert DNA were chosen. Molar ratios were calculated using the following equation:

Amount of vector x size of insert x molar ratio of i nsert = amount of insert (ng)

Size of vector (kb) vector

2.2.7 Addition of Iinkers

Not I (New England Biolabs) and Not I-Eco RI (Pharmacia B iotech) linkers were ligated to blunt-ended DNA fragments as described by Sambrook et al. ( 1 989). Up to 0.5 Ilg of blunt-ended DNA, 2 Ilg of phosphorylated Not I or Eco RI-Not I linker, 10 JlL of 2 x rapid ligation buffer and 10 units of T4 DNA ligase (Roche) were mixed in a final volume of 20 ilL and incubated at ambient room temperature for 5 min. Following ligation, the resulting fragment was digested with the appropriate restriction enzyme as described in Section 2.2.2, and cloned into the appropriate restriction site of the selected vector.

2.2.8 Dephosphorylation of Iinearised plasmid DNA

Linearised plasmid DNA was dephosphorylated using ,S.hrimp Alkaline Ehosphatase (SAP) to remove 5' phosphates (Roche) as per the manufacturer' s instructions. Up to 1 pmol 5'-terminal phosphorylated DNA fragments either 5' protruding or 5' recessive ends were incubated with 1 unit of SAP at 37°C for 10 min. Up to 0.2 pmol 5'-terminal phosphorylated DNA fragments blunt-ended were incubated with 1 unit of SAP at 37°C for 60 min. Following dephosphorylation reactions, SAP was deactivated by incubation at 65°C for 1 5 min, the dephosphorylated fragments were then used without further

Chapter 2, Methods and Materials

purification for ligation reactions. Dephosphorylation of plasmid vectors prior to ligation reactions ensures self-ligation of the vector occurs at very low frequency.

2.2.9 Bacterial culture

Media used:

51

• LS - 1 0 9 L·1 bacto-tryptone, 5 9 L·1 yeast extract, 1 0 9 L·1 NaCl, for solid media 1 .5% (w/v) bactoagar was added

• YES - 1 9 L·1 yeast extract, 5g L·1 beef extract (Sovril), 0.5 9 L·1 bacto-peptone, 0.5 9 L·1

sucrose, 0.5 9 L·1 MgS04.7H20, pH 7.0, for solid media 1 .5% (w/v) bactoagar was added

E. coli strains were grown on/in LB or liquid medium at 37°C under appropriate antibiotic selection. A. tumefaciens was grown on/in either LB or YEB or liquid medium at 28°C under appropriate antibiotic selection. B acterial cultures grown in liquid medium were aerated by constant orbital shaking at 240-300 rpm.

2.2.1 0 Bacterial transformation Media used and reagents:

• LS - 1 0 9 L·1 bacto-tryptone, 5 9 L·1 yeast extract, 1 0 9 L·1 NaCI

• YES - 1 9 L·1 yeast extract, S 9 L·1 beef extract (Sovril), 0.5 9 L·1 bacto-peptone, 0.5 9 L·1

sucrose, 0.5 9 L·1 MgS04.7H20, pH 7.0

• SEM buffer - 1 0 mM piperazine-NN-bis-2-ethanesulphonic acid (PI PES), 55 mM MnCI2, 1 5 m M CaCI2, 250 mM KCI, p H 6.7

2.2.1 0.1 Competent cells Competent E. coli cells for heat shock transformation were prepared as described by Sambrook et al. ( 1 989) or purchased (DH5a, Invitrogen LIFE TECHNOLOGIES). An overnight culture ( 1 00 mL) was grown in liquid LB medium at room temperature (ca. 22°C), or to an OD600 0.4-0.8 (0.6 optimal). Cells were chilled on ice for 20 min, followed by harvesting by centrifugation at 3000 x g at 4°C for 1 0 min. The cells were then resuspended in 20 mL ice-cold filter sterilised SEM buffer. The resuspended cells were incubated on ice for 5 min, followed by centrifugation at 3000 x g for 10 min. The cells were resuspended in 6 mL ice cold filter sterilised SEM buffer and 460 ilL DMSO was added. The cells were divided into 200 ilL aliquots, snap frozen in liquid nitrogen and stored at -70°C until required.

Chapter 2, Methods and Materials ₅₂

Competent A. tumefaciens cells for electroporation were prepared as described by Sambrook et al. ( 1 989), or purchased (LBA4404, Invitrogen, LIFE TECHNOLOGIES). A culture (250 mL) was grown in YEB media at 28°C until an OD6oo of between 0.5-0.7 was reached. The cells were harvested by centrifugation at 3000 x g at 4°C for 10 min. The cells were resuspended in 250 mL of cold 10% glycerol. The cells were again pelleted as above and resuspended in 200 mL ice cold 10% glycerol. The process was repeated as above with reduction in volume continuously from 1 50 mL, 1 00 mL, 50 mL

to finally 500 �L 10% glycerol. Cells were divided into 40 �L aliquots and stored at - 70°C until required.

2.2.1 0.2 Transformation E. coli strains DH5a (Invitrogen, LIFE TECHNOLOGIES) or XL- I blue (Stratagene) were transformed by heat shock at 42°C (Sambrook et al. 1989). Competent cells (50- 100 �L) stored at -70°C were thawed on ice, plasmid DNA or ligation reaction mix were then added, and left to incubate on ice for 30 min.

Subsequently, the cells were heat shocked at 42°C for 1 min, and returned to ice for 2 min. Cells were then left to recover with the addition of 900-950 �L L-Broth at 37°C for at least 1 h followed by plating on appropriate selective media. For transformation of E.

coli cells using l igation reactions, often blue/white colour screening was used to

determine which cells contained the inserted DNA fragment, by addition of X-gal and IPTG to the surface of selective media (Sambrook et al. 1989). The pUC based vectors pBLUESCRIPT® (Stratagene), pGEM®-5Zf, pGEM®-7Zf, pGEM®-T Easy (Promega), and binary vectors pART27 (Gleave 1 992) and pBJ49 used in this work all have the gene encoding the lacZ a-peptide, allowing recombinants to be selected by blue/white colour screening.

A. tumefaciens strains LBA4404, GV3 101, and AGL l were transformed by

electroporation. Competent cells stored at -70°C were thawed on ice. Plasmid DNA ( 1 �L) was combined with 20 � of competent cells and placed i n an electroporation cell (Gene Pulser® Cuvette, BioRad) and the cells electroporated with a 12 KV cm-1 charge (Cell Porator BRL LIFE TECHNOLOGIES). Immediately following electroporation the cells were recovered by the addition of 1 mL of TB (terrific broth) and shaken at 28°C for 2-4 hours. The cells were then plated on solid agar media with appropriate antibiotic selection and incubated at 28°C for three days.

Chapter 2. Methods and Materials ₅₃