Estrategias

4.1.2.1 Diagnostic and preparative use of restriction endonucleases.

Restriction endonucleases were supplied by their manufacturers with the appropriate buffers and were used according to manufacturers' instructions. The progress o f each reaction was assessed by agarose gel electrophoresis as described in section 4.1.3.1. Partial digests were assessed by taking samples from the reaction at various times until completion and comparing the reaction products to the uncut plasmid. For full digests, completion of the reaction was verified by ensuring the absence of uncut plasmid bands in each lane. DNA markers (lkb ladder, Gibco BRL) were always used as a reference. In the case of double digests, the route chosen to achieve the desired reaction depended upon the properties of the enzymes involved. If both enzymes worked with greater than 80% efficiency in the same buffer at the same temperature, the reactions were carried out concurrently. If both enzymes worked with greater than 80% efficiency in the same buffer, but the optimal temperature varied, the reactions were carried out consecutively with an intermediate heat inactivation step if necessary. If the enzymes could not work with greater than 80% efficiency in the same buffer, but one buffer could be converted into the other e.g. by increasing the salt concentration, the reactions were carried out consecutively with the necessary supplementation step and an intervening heat inactivation step if necessary. If the enzymes were totally incompatible, and no route could be found to combine them, the products of the first digest were purified using the Geneclean II kit according to the manufacturer's instructions and redissolved in the buffer appropriate for the subsequent reaction.

4.1.2.2 Filling recessed 3' ends with the Klenow fragment of E. coli DNA polymerase I

To facilitate the joining together of incompatible sticky ended DNA fragments, the recessed 3' ends of certain restriction digestion products were filled using the 5'->3' polymerase activity of the Klenow fragment of E. coli DNA polymerase I. The procedure followed was similar to that presented in Sambrook et al. (1989), appendix

M aterials a n d M ethods: C hapter 4

F2-F3, although the final concentration of each dNTP in the reaction was 1mA/, not 0.5mA/ as suggested.

4.1.2.3 Removing overhanging 3' ends with T4 DNA polymerase

Recessed 5' termini cannot be filled in the same manner as recessed 3' termini because of the polarity of all DNA polymerases and their inability to initiate DNA synthesis de novo. To generate blunt ends from overhanging 3' termini the 3'=>5' exonuclease activity of bacteriophage T4 DNA polymerase was exploited. This enzyme is more suitable than the Klenow fragment for this particular application because it's exonuclease activity is 2-300 fold greater than that of the other enzyme. Whilst trimming overhanging 3' termini, the enzyme simultaneously fills in recessed 3' termini.. The Klenow fragment of DNA polymerase I was chosen when end-filling alone was required simply on the basis of its cost. The procedure followed was similar to that presented in Sambrook et al. (1989), appendix F4-F5, although the final concentration o f each dNTP in the reaction was 0.1 mA/, not 2mA/ as suggested.

4.1.2.4 Removing 5'-terminal phosphate groups with calf intestinal alkaline phosphatase

The removal of 5'-terminal phosphate groups from linearised plasmids with

compatible ends helps to limit intramolecular ligation. The method used was similar to that presented in Sambrook et al. (1989), pp 1.60-1.61, although the same amount of enzyme (1 unit per 2 pmoles 5'-terminal phosphate residues) was used for both protruding 5' termini and blunt or recessed 5' termini, resulting in a great excess of enzyme for the former reaction. Following restriction digestion, 1 Ox

dephosphorylation buffer (supplied by the manufacturer) and enzyme were added directly to the reaction mixture with no intermediate phenol :chloroform extraction and ethanol precipitation step which is advised in the method of Sambrook and

colleagues. A phenolxhloroform extraction step followed by ethanol precipitation was carried out after the reaction was complete in order to remove all traces o f the enzyme. This was necessary because even small amounts of phosphatase activity in the subsequent ligation reaction could dramatically reduce its efficiency.

M aterials a n d M ethods: Chapter 4

4.1.2.5 Ligation

Ligations (involving cohesive and blunt-ended fragments) were carried out using bacteriophage T4 ligase according to the method of Sambrook et al. (1989), pp 1.68-

1.70. Bacteriophage T4 ligase buffer was supplied by the manufacturer as a 5x concentrate and was used according to manufacturer's instructions. For the majority of ligations, a recombinant plasmid containing an insert o f foreign DNA was generated. Under these circumstances, a threefold molar excess of insert over vector fragments was used in the reaction and the vector fragment was pretreated with CIAP to limit intramolecular ligation (see section 4.1.2.4). No condensing agents were used in blunt-ended ligation reactions, but the concentration o f vector fragments was increased from lOpg m b1 to lOOpg mH with a proportionate increase in the

concentration of insert. The concentration of bacteriophage T4 DNA ligase used was 50 Weiss units mb' (Weiss et al., 1968) for both cohesive and blunt-ended ligations. To verify the various stages of the subcloning procedure, several control ligations were carried out in parallel as shown in table 4.1. These controls were still carried out if subcloning was directional (i.e. the vector ends were incompatible) as they then gave some indication o f the performance of the restriction endonuclease digestions.

Control Contents Purpose

A Dephosphorylated vector

No insert

Tests for success o f dephosphorylation

B Untreated vector

No insert

Tests for integrity of vector sequence and indicates maximum transformation efficiency

C No vector

Untreated insert

Tests for purity of insert

D No DNA Tests for purity of reaction components and

integrity of bacteria Table 4.1: Controls used to verify ligation reactions

4.1.2.6 Transformation of competent E scherichia c o li cells with plasmid vectors

Competent Escherichia coli cells were prepared freshly for each transformation according to the method of Cohen et al. (1972). An additional step was included in which the cells were resuspended in cold 1 OOmM MgCl2 then pelleted at 2000 rpm prior to resuspension in cold 100mA/ CaCl2 as described in the published method. This step was reported to increase the transformation efficiency (C. Mason, pers. comm.). Transformation was carried out with 200pl competent cells using approximately 50ng DNA (from a ligation reaction or freshly diluted from plasmid stock) according to the method of Cohen et al (1972). All plasmids used in this study

M aterials and M ethods: C hapter 4

conferred ampicillin resistance upon their hosts and it was therefore unnecessary to allow the cells to recover following transformation. Transformed cells were plated on LB-agar supplemented with lOOpg ml-1 ampicillin, incubated at 37°C overnight and transferred to 4°C before satellite colonies were able to grow.