The bacterial strain used was Escherichia coli K12 DH5 alpha F . The plasmid vectors used were :
pSP64TK l (Xu e ta l, 1995). pEluescript KS +/- or SK +/-
2.2.1 Production of competent cells for transformation.
K12 DH5 alpha F' were streaked on an L-agar plate and grown overnight at 37°C. A single
colony was used to inoculate 10ml L-Broth for an overnight culture at 37°C with shaking. The following day 50ml of L-Broth was inoculated with 0.5ml of culture and grown until the OD5 5 was 0.45- 0.55. The culture was then chilled on ice for 10 minutes and spun at
13,000rpm for 10 minutes to pellet the cells. The supernatant was discarded and the pellet was resuspended in 20ml TfBI and incubated on ice for 5 minutes. The suspension was spun as before and the resulting pellet was resuspended in 2ml TfBII and again incubated on ice but this time for 10 minutes. The cells were snap frozen in aliquots of lOOpl on a
mixture of dry ice and ethanol and stored at -70°C until required.
2.2.2 Subcloning and ligation.
The T4 DNA ligase enzyme and buffer were used according to the manufactures instructions with the ratio of vector to insert being 1:3. The reaction was either performed overnight at 16°C for blunt ended ligations or for a few hours at room temperature for cohesive ended ligations.
2.2.3 Bacterial transformation.
3pl of a ligation reaction was added to 50|il of competent cells which had been allowed to defrost slowly on ice. This mixture was incubated on ice for 15 minutes then given a brief
heat shock by placing in a 42°C waterbath for 90 seconds. The cells were then incubated
for a further 2 minutes on ice before adding 200pl of L Broth. The cells were allowed to
recover at 37°C for 50 minutes and then plated out on L agar plates containing lOOpg/ml
ampicillin. The plates were incubated overnight at 37°C and single colonies were picked for further analysis.
2.2.4 Isolation of plasmid DNA by a mini-preparation method.
A single bacterial colony was used to inoculate a 5ml culture of L-Broth containing lOOjX
g/ml ampicillin, which was incubated overnight at 37°C with shaking. 4ml of the culture
were spun down at 13,000 rpm using sterile 2ml Eppendorf tubes, saving 1 ml for possible future analysis. The pellet obtained was resuspended in 180|il of Solution One and allowed
to sit at room temp for 5 minutes and then on ice for 5 minutes. 400|il of 0.2M NaOH, 0.1% SDS was added to the mixture before being mixed gently by hand and allowed to remain on ice for a further 5 minutes. Next 300pl of 3M NaOAc was added and the tube
was mixed gently. The tubes were put on ice for 10 minutes and subsequently spun for 15 minutes at 4°C at 13,000 rpm. 750pl of the supernatant was transferred to fresh 2ml
Eppendorf tube, where 450|il of isopropanol was added and the resulting mixture was vortexed. The tubes were spun for 5 minutes at room temperature and the pellet was washed in 70% EtOH and air-dried before resuspension in lOOpl of double distilled H2O.
At this stage diagnostic digests were carried out to analyse the DNA construct.
2.2.5 Isolation of plasmid DNA using the Wizard Maxiprep DNA purification system.
100-500ml of L-Broth containing lOOpg/ml ampicillin were inoculated with 1ml of a
desired culture and shaken overnight at 37°C. The plasmid DNA was subsequently purified
using the wizard maxiprep DNA purification system (Promega A7270). Diagnostic digests were carried to authenticate the DNA.
2.2.6 R estriction endonuclease digests.
All restriction enzymes were used according to the manufacturers instructions, using the appropriate buffer supplied.
2.2.7 Electrophoretic separation of DNA and RNA.
DNA or RNA samples were analysed using agarose gel electrophoresis in TBE buffer. The concentration of agarose used depended on the size of the fragments that were to be separated but generally 1% gels were used. DNA or RNA samples were loaded after mixing with one tenth volume of orange loading buffer. Fragment sizes were determined by corunning a 1 Kb ladder and ethidium bromide stained DNA or RNA was visualised using a UV transilluminator and photographed.
2.2.8 Polymerase Chain Reaction analysis.
A 25 base pair oligonucleotide was designed to check the orientation of the Elk insert being subcloned into pBluescript. The conditions were 94°C for 1 minute, 57°C for 1 minute,
then 72°C for 2 minutes for 25 cycles. A fraction of each reaction was then loaded on an agarose gel to determine which reaction yielded a product.
2.2.9 In vitro transcription and translation.
cDNA was transcribed and translated using the coupled transcription-translation rabbit reticulolysate system (Promega) labelling with ^^S-methionine. The products were heated at 95°C for 2 minutes and separated on a 1% SDS PAGE gel using rainbow markers (Amersham) as size standards.
In vitro transcription of synthetic mRNA was achieved by mixing the following at room temperature: 35.5|il DEPC H^O, 20pl 5x transcription buffer, 12.5|il lOOmM DTT, lOpl
lOx nucleotide mix, lOjxl lOmM GpppG, 4\i\ RNAsin, 4|Lil SP6 RNA polymerase, 4|il
linearised plasmid (Ipg/pl) and incubated at 37°C for 2.5 hours. Ijil aliquot was removed
and frozen before adding 2\i\ of RNAse-free DNAse (Promega M6102) and the tube further incubating at 37°C for 20 minutes.
Synthetic mRNA was then purified by phenol/chloroform and chloroform extraction, followed by the use of a Quick Spin Column (Boehringer Mannheim 1274015) and
subsequent precipitation at -20°C with 1/3 volume lOM ammonium acetate and 2.5
volumes of ethanol. The RNA was pelleted by centrifugation at 13,000rpm, 4°C for 20 minutes before being washed in 70% ethanol and air-dried. The RNA pellet was dissolved in ultrapure water obtained from Sigma (W4502) and a Ipl aliquot was run adjacent to l|il aliquot kept prior to DNAse treatment on a 1 % TBE RNAse-free gel to estimate the final concentration of RNA.