Materials and methods 1 0

Isolation of DNA fragments

DNA fragments were excised from agarose gels and DNA was isolated using the Geneclean II® kit (BIO 101 Inc., CA) according to the manufacturers instructions.

Ligation of DNA fragments

To ligate DNA fragments into a cut plasmid T4 DNA ligase was used which catalyses the formation of phosphodiester bonds between neighbouring 3'-hydroxyl and 5'-phosphate ends in double stranded DNA. Typically a 3 molar excess of cDNA fragment compared to linear plasmid was used, keeping the total amount of DNA to less than 50 ng. Ligation was performed in a volume of 10 pi in 1 x T4 DNA ligase buffer (GIBCO) using 1 U T4 ligase (GIBCO) for 1 h to 2 h at room temperature or alternatively for 8 h to 15 h at 16 °C.

DNA transformation of E. coli

The ligation reaction (less than 50 ng DNA in 10 pi volume) or 0.5 ng to 1 ng of other circular plasmid DNA was added to 20 pi to 100 pi of competent E. coli cells (see below) on ice and mixed gently. After 30 min on ice, the E. coli cells were heat shocked by placing for 5 min at 37 °C (or for 2 min at 42 °C) and then returned to ice. 200 pi of L- broth was added and cells were left to recover for 30 min to 60 min at 37 °C . Subsequently cells were plated onto L-broth agar plate containing typically 50 pg/ml ampicillin.

Preparation of competent bacteria ceiis

E. co//strains used were JM101 or DH5 for amplification of plasmid DNA and XLI-Blue for expression of recombinant proteins. Best results for preparation of plasmid DNA have been observed with the DH5 E. coli strain. A colony was picked from a minimal plate and grown overnight in 25 ml L-broth. The culture was diluted 1/200 in 400 ml L-broth and E. co//were grown to logarithmic phase (OD5 5o=0.4 5). The cells were

Chapter 2 Materials and methods 1 0 1

chilled on ice, spun at 4,000 rpm for 10 min at 4 °C and resuspended in ice-cold 160 ml

transformation buffer I {30 mM potassium acetate, 100 mM RbCIa, 10 mM CaCl2, 50 mM

MnCl2, 15% glycerol; final pH5.8). After 30 min on ice, bacteria cells were pelleted at

4,000 rpm for 10 min at 4 °C, the cells were resuspended in 32 ml transformation buffer I!

(10 mM PIPES, 75 mM CaCl2, 10 mM RbCl2, 15% glycerol; final pH6.5) and incubated

for 30 min on ice. Aliquots of 400 |xl were snap frozen on cardice and stored at -70 °C.

Mutagenesis of DNA using poiymerase chain reaction

Polymerase chain reaction (PCR) was employed to change sequences close to the ends of a cDNA fragment using the Taq polymerase which is a highly processive 5'- 3' DNA polymerase lacking 3'-5' exonuclease activity, originally from Thermus acquaticus

strains. An oligonucleotide in sense orientation corresponding to the 5' end of the cDNA fragment was used together with an oligonucleotide in the antisense orientation corresponding to the 3' end. Reactions were performed in a total volume of 100 \l\

containing 100 ng DNA template, 2 p.1 dNTP mix (dATP, dCTP, dGTP, dTTP; each 10 mM), 10 111 10 X PCR reaction buffer (100 mM Tris pH8.3, 25 mM MgCl2, 500 mM KCI,

0.01% gelatine), and 5 |il 3' and 5' primer each (stock conc. each 5 |liM ). 2 units (0.4 p.1) of Taq polymerase (Perkin Elmer) were used per reaction and the sample was overlaid with mineral oil (Sigma) to prevent evaporation. Typically an initial 96 °C melting step was followed by 35 cycles of 94 °C 1 min, 2 min annealing at 60 °C and 2.5 min extension at 72 °C. A final 9.9 min chase step at 72 °C was included. To test for the size of the PCR product 10 |liI sample were loaded on an analytical agarose gel. The PCR products in the remaining sample were removed from primers and unincorporated nucleotides using the Geneclean II® kit (BIO 101 Inc., CA). The PCR products were used for cloning after phenol/chloroform extraction and ethanol precipitation of the sample.

Chapter 2 Materials and methods 1 0 2

Sequencing of DNA

A standard single stranded DNA sequencing protocol was applied which is based on the dideoxy chain termination principle of Sanger using the Sequenase® version 2.0 17 DNA polymerase kit (United States Biochemical). The radioactive labelling of the DNA was accomplished by adding ^^s-labelled dATP (Amersham). To denature the double stranded plasmid DNA, 2 pg plasmid DNA resuspended in 18 pi water were mixed with 2 pi of 2 M NaOH and incubated for 15 min at 37 °C. The denatured DNA was precipitated quickly with 100 pi ethanol in the presence of 4 pi 10 M NH4AC. After 20 min on ice the DNA was pelleted in a microcentrifuge at 15,000 x g fo r

10 min at 4 ^0, the DNA pellet was washed with 75% ethanol, dried and resuspended in 7 pi water. 2 pi of 5 x Sequenase reaction buffer and 1 pi of primer (stock conc. 5

pmol) was added and the primer was allowed to anneal to the DNA template for 20 min at 37 °C. The sample was chilled on ice and sequencing reactions were carried out according to the manufacturers instructions.

Samples were loaded onto a 6% or 8% polyacrylamide gel , containing 7 M

urea in 1 x TBE buffer {89 mM Tris-borate, 89 mM boric acid, 2.5 mM EDTA; final pH8.3; 10 X stock: 108 g Tris, 55 g boric acid, 10 ml of 0.5 M EDTA pH8 , ad II) and

electrophoresis was carried out for about 2 h at 38 W. The gel was fixed in 1 0%

methanol/10% acetic acid for 10 to 15 min and dried under vacuum at 80 °C . Autoradiography was performed overnight at room temperature.