2.1.1 DNA Constructs
HRP-P-selectin is a chimera comprising the human growth hormone signal sequence, followed by horseradish peroxidase (HRP) and the transmembrane (TM) and cytoplasmic domains of P-selectin in pRK34 (Norcott et al. 1996).
763|_|Rp_p_seiectin is a truncation of the cytoplasmic tail (Blagoveshchenskaya et al. 1998a), and °"^^^HRP-P-selectin are tetra-alanine mutations as
described (Blagoveshchenskaya et al. 1998b).
Wild-type GFP-CD63 in the pEGFP.C2 vector and a series of single amino acid substitutions in the sequence GYEVM in the cytoplasmic tail of CD63 in pMEP4 were kind gifts from Paul Luzio, Laboratory for Molecular Biology, Cambridge University.
2.1.2 Agarose Gel Electrophoresis
1% agarose (Gibco) in IxTAE (40mM Tris-acetate, ImM EDTA) was dissolved by heating, and when cool, ethidium bromide was added to the solution (0.4|Lig/ml) and the gel poured. DNA samples were mixed with 6x loading dye (Promega), loaded onto the gel along with a Ikb ladder (Promega) and resolved by running at 100V for ~1 hour. DNA was visualised with a UV transilluminator.
2.1.3 Restriction Digests
0.5-1 ^ig of plasmid DNA was digested in a total volume of 20)liI with 2pl each of the
appropriate enzymes, 2|xl of the corresponding buffer and Bovine Serum Albumin (BSA) for 2 hours at 37°C.
2.1.4 Fragment Purification
Digested DNA was resolved by agarose gel electrophoresis and the appropriate band was excised using a clean scalpel blade. DNA fragments were purified using the Quiagen Gel Extraction Kit according to the manufacturers instructions. DNA was eluted in a volume of 30^1.
2.1.5 Ligations
In a total volume of lO^il, a ratio of 2:5 vectoriinsert was ligated with lp.1 T4 DNA ligase (Promega) in ligase buffer. For control ligations, 2\i\ of vector and no insert was used. Ligation mixes were left overnight at 4°C and the reaction then stopped by heating to 70°C for 4 minutes.
2.1.6 Transformation of Competent Cells
DH5aMRC™ (Gibco) competent cells were used. Briefly, 50^1 competent cells were mixed with 5\i\ of ligation mix, incubated on ice for 30 minutes and then transferred to 42°C for 45 seconds. 450)liI LB (per litre: lOg tryptone, 5g yeast extract, lOg NaCI) was then added and incubated at 37°C for 1 hour with shaking (225rpm). Bacteria were then spread on Kanamycin (25ng/ml) LB agar (15g/litre agarose) plates and incubated at 37°C overnight. As a negative control, vector only ligation, and as a positive control, wild-type GFP-CD63 were used to transform bacteria.
2.1.7 DNA Recoverv
Mini-preps Five colonies per transformation were picked and grown up in 2ml LB
Kanamycin (25|Lig/ml) for 5 hours at 37°C with constant shaking at 225rpm. The Stratagene Mini-Prep Kit was used to extract and purify DNA from 1.5ml of the culture to give 50|il DNA in IE (lOmM TrisCI pH8.0, ImM EDTA). These DNA samples were verified by restriction digestion using the same enzymes that were used to prepare the fragments before ligation.
Maxi-preps: alkaline lysis 50^1 of mini-prep culture was added to 100ml LB Kanamycin (25|uig/ml) and incubated overnight at 37°C with constant shaking. Bacteria were spun down at 4,000rpm for 10 minutes and the pellet resuspended
in 25ml STE (10mM TrisCI pH8.0, 0.1M NaCI, 1mM EDTA) and pelleted once more. 18ml of Solution I (50mM glucose, 25mM TrisCI pH8.0, lOmM EDTA) was used to resuspend the bacterial pellet, to which 40ml of Solution II (0.2M NaOH, 1% SDS) was then added. Following 10 minutes of gentle mixing at room
temperature, 20ml of ice-cold Solution III (60ml 5M KAc, 11.5ml glacial acetic acid, 28.5ml H2O) was added and left on ice for 10 minutes. The resulting bacterial lysate was centrifuged for 30 minutes at 4,000rpm. The supernatant was passed through glass wool and 0.6 volumes of isopropanol added, mixed and left for 10 minutes at room temperature. Precipitated nucleic acids were recovered by
centrifugation for 15 minutes at room temperature. The resulting pellet was ethanol washed and resuspended in 3ml TE.
2.1.8 DNA Purification
3ml of maxi-prep DNA was weighed and 1.01 g solid CsCI per gram of DNA added and warmed to 30°C to dissolve. lOOpI of lOmg/ml ethidium bromide for each 5g of maxi-prep DNA was added and the precipitate removed by centrifugation for 5 minutes. The clear red solution was then subjected to equilibrium gradient centrifugation at 100,000rpm overnight at 20°C. The lower band of plasmid DNA was transferred and the ethidium bromide removed by butanol extraction. CsCI was removed from the solution by ethanol precipitation. Briefly, 3 volumes of H2O were added to dilute the CsCI, and 8 volumes of ethanol. This solution was mixed and left at 4°C overnight. The DNA precipitate was recovered by centrifugation at 20,000g for 15 minutes. Following a 70% ethanol wash, the DNA was finally resuspended in 1ml TE. The DNA concentration was determined by reading the absorbance in a GeneQuant pro.
2.1.9 DNA Sequencing
Oligonucleotides were synthesised by and sequencing reactions carried out by Alta Bioscience (School of Biosciences, University of Birmingham). For automated sequencing, forward and reverse oligonucleotides were designed to the pEGFP.C2 vector upstream and downstream of the inserted CD63 sequence.
R: reverse 22 bases 5'-TCA.GGG.GGA.GGT.GTG.GGA.GGTT-3' These were made up to a final concentration of 50mM. For sequencing, l^ig of plasmid DNA and 90pmol of each primer were used.