Resultado de la Encuesta a Dueños de Procesos

2.15.1 Features of the Phagemid

All DNA amplified by PCR was cloned into the phagemid pBluescript KS* purchased from Stratagene (Cambridge, UK.), figure 2.1. This is a 2958 base pair phagemid derived from pUC19 which contains an ampicillin resistance gene for antibiotic selection. The LacZ gene allows the expression of the a-peptide of P-galactosidase which can be used for blue/white colour selection. When the multiple cloning site (MCS) is interrupted this prevents expression of the peptide and the colonies remain white.

2.15.2 Restriction Endonuclease Digestion

The restriction endonucleases SamHI, Hind\\\, Psfl, and Sa/I were used in conjunction with the REact buffer system, all from Life Technologies (Paisley, UK ). The manufacturer provided the appropriate REact buffer (nos. 1-11) for each restriction enzyme. In situations where a double digest was performed and the recommended choice of buffers was different for each enzyme, a compromise buffer was chosen with respect to salt concentration which provided the optimum activity possible for each enzyme. The most frequently used REact enzymes are given in table 2.5.

NaeI 134 Ssp I 445 S s p l2 8 5 0 N a e I 333 Xmn I 2645 Sea I 2526 Pvu I 503 . Pvu II 532 Pvu I 2416 Kpn I 657

Bluescript® SK +/-

Figure 2.1 The 2958 base pair phagemid Bluescript. This vector is derived from pUC19. The multiple cloning site is denoted as MCS, in the polylinker shown here L a cZ transcription proceeds from the Sac! to Kpn\ restriction enzyme sites. LacZ transcription proceeded in the opposite direct in the

Table 2.5

The buffers used for restriction digests

Buffer Enzyme Concentration

REact 2 H/ndlll/Psfl SOmM TrisHCI pHS.0,1GmM MgClj, 50mM NaCI REact 3 BamH\ 50mM TrisHCI pH8.0,1GmM MgClj, 1GGmM NaCI REact 10 Sal\ IGGmM TrisHCI pH7.6, IGmM MgClj, ISGmM NaCI

The restriction endonucleases described above were used to prepare PCR products for cloning into similarly digested pBluescript KS*. The 'Gene Cleaned' PCR product and vector were digested in parallel at 37®C for 1 hour Digestion volumes were up to 20pl, with the combination of restriction enzymes accounting for not more than 10% of the total volume in 10% REact buffer. Impurities were removed from the digested vector and insert DNA by Gene Clean, and the digestion checked by agarose gel electrophoresis.

2.15.3 Ligation of PCR Product Into pBluescript KS*

Digested PCR products were ligated into pBluescript KS* using T4 DNA ligase. The ligase was obtained from Amersham International (Amersham, UK.) and used with a 5 x reaction buffer (Life Technologies. Paisley, UK ). Ligation of 100-200ng of vector DNA took place with a three fold excess of the PCR product insert.

2.15.4 Preparation of Competent Cells

Competent cells were prepared using XL-1 Blue E.coli (Stratagene, Cambridge, UK ), using the method of Cohen et al (1972). Agar was prepared by adding 15g/l BactoAgar to LB medium. The agar was heated until it dissolved

and ampicillin (100pg/ml) was added. 25ml of agar was poured into each plate, (Nunc, Life Technologies. Paisley, UK.), and allowed to cool. E. co// were plated for single colonies using an inoculating loop and incubated overnight at 37**C. A single colony of the plated XL-1 blue E. co// was selected and added to 10ml LB medium containing 12.5pg/ml tetracycline in a 30ml sterilin tube and cultured overnight at 37®C in a shaking incubator (220rpm). 1ml of this starter culture was used to inoculate 100ml of LB medium in a 1 litre flask. The culture was incubated at 37*C with shaking (220rpm) until the absorbance at GOOnm had reached 0.2-0.3 (approximately 2 hours) and then placed on ice for lOmin. 80ml of the culture was centrifuged at 4®C in a MSB Mistral 2000 at 4,000 x g for lOmin. The pellet was resuspended in ice cold 0.1ml 0.1 M calcium chloride, left on ice for lOmin and then recentrifuged. The pellet was washed twice and finally resuspended in a total volume of 1ml ice cold 0.1M calcium chloride. The cells were kept on ice for at least 2 hours before use.

2.15.5 Transformation

Competent cells (75pl) were added to each ligation reaction. Transformation of competent cells with uncut pBluescript KS* and pBluescript KS* that had been digested with the appropriate restriction enzyme, but had not been religated, were used as controls. Competent cells and ligation mixture were left on ice for 30min and then placed at 42*"C for 45 seconds. 300pl of LB medium, prewarmed to 37®C, were added and the cells incubated for 50min at 37'^C and 2 2 0rpm to allow the cells to recover and begin to express antibiotic resistance. 25pl of transformed cells were mixed with, 40pl X-gal, 40pl lOOmM IPTG (for blue/white colour section) and 175pl LB medium and plated out, one plate per cloned product, on LB agar containing ampicillin (50pg/ml). The plates were incubated overnight at 37®C for a maximum of 16 hours. The number of colonies on each plate was counted so that the efficiency of transformation could be calculated. White colonies (10-20) were selected and grown overnight in 5ml LB medium containing ampicillin (100pg/ml) at 37®C and 220rpm. These

cultures were used to prepare plasmid DNA in order to check for correct ligation and transformation.

2.15.6 Plasmid Preparation from Ceiis

Plasmids were prepared from overnight cultures using the 'Magic' and 'W izard Miniprep' plasmid preparation kits from Promega Corporation (Southampton, UK.). Briefly, 3ml of overnight culture were pelleted by centrifugation. The cells were resuspended and lysed using the manufacturer's resuspension and lysis buffers in a total of 400pl. Cell debris was removed by centrifugation (Eppendorf centrifuge at 13,000rpm for 30 seconds). Plasmid DNA present in the supernatant was bound to the 'Miniprep' column (supplied by the manufacturer). The columns were washed with 3ml ethanol solution (50% v/v) and the bound plasmid DNA eluted from the column in 20pl DEPC treated water by centrifugation (Eppendorf centrifuge at 13,000rpm for 30 seconds) into an Eppendorf tube.

The inserted DNA was cut out of the pbluescript KS* using the appropriate restriction enzymes (as described earlier) and checked for size by electrophoresis. The remainder of the plasmid prepared was stored at -20®C.