8.2.1 Preparation of competent bacteria
250mls of LB w as inoculated w ith 10-12 fresh colonies of D H 5a. The culture was incubated at 18°C w ith vigorous shaking until 00^00=0.6. The bacteria were harvested by centrifugation at 2500g for lOmins at 4°C, w ashed once w ith ice cold TB pH6.7 (lOmM pipes, 55mM MnCl2,15m M CaCl2, 250mM
KCl), then resuspended in 20mls TB. DMSG was ad d ed slowly to a final concentration of 7% while gently swirling to mix. 400jil aliquots were frozen on dry ice, and stored under liquid nitrogen.
8.2.2 Chemical transformation of bacteria
5|il of ligation, or Ijil of plasm id (O.l-lOOng) was placed in a microfuge tube on ice. 200|il of thaw ed celbw ere added, and mixed w ith the DNA by gentle flicking. The sam ple was incubated on ice for 30min, heat shocked at 42°C for 30secs, then placed back on ice. 800|il of BHI was ad d ed and the sam ple incubated at 37“C for 1 hour. A 200pl aliquot was spread on an LB agar
plate containing lOOpg/pl ampicillin. The plate w as incubated overnight at 37“C.
8.2.3 Preparation of electrocompetent bacteria
Bacteria were streaked from a glycerol stock onto an LB plate and grow n overnight at 37°C. A single fresh colony was used to inoculate a 5ml preculture w hich was grow n to saturation overnight. 200mls of LB was inoculated w ith 200|il of the preculture. The culture was grow n to an OD5 9 5 of 0.5 w ith
vigorous shaking, then chilled rapidly on ice. The bacteria were harvested by centrifugation at 3000g for 5mins, the supernatant was rem oved and the pellet w ashed twice w ith ice cold Im M hepes pH7.5, before resuspension in 2mls ice cold water.
2.4 Electroporation of bacteria
40jil of thaw ed cells were added to a prechilled 2mm gap electroporation cuvette. The DNA sam ple (5pl of ligation, or ljj.1 of plasm id (O.l-lOOng) was a d d e d to the cells and m ixed by gentle pipetting. E lectroporation w as perform ed using a 'Gene-Pulser' (Biorad) set at 2000, 25uF and 2250V. 1 ml of BHI w as added to the cuvette immediately after electroporation, and the cells left to recover for 1 hour at 37°C. A 200|il aliquot was plated onto LB agar plate containing lOOjig/ml ampicillin. The plate was then incubated overnight at 37° C.
2.5 Preparation of plasm id DNA
The majority of plasm id preparations were perform ed using an A utogen 740 p la s m id p r e p a ra tio n m ach in e. P la sm id s u se d for in vitro transcription/translation, were prepared using a Quiagen m axiprep kit.
8.2.6 Restriction digestion
Quick digests to check constructs were perform ed in a 20|il volum e in a m icrotitre using 5 units of restriction enzym e in the a p p ro p riate buffer (Boehringer) and w ith an incubation time of 30mins-l hour. For digestion of vectors for use in sub cloning, approximately Ip g of vector was digested for 3- 16 hours w ith approxim ately 20 units of enzym e in a volum e of 50jil. W hen digesting of the ends of PCR products, the PCR product was first gel purified.
8.2.7 Ph^phatasing vectors
1 0 units of calf intestinal alkaline phosphatase were added directly to the
restriction digest after the incubation period. The reaction was then incubated for a further 3-16 hours.
8.2.8 Ligation reactions
All ligation reactions were perform ed in a volume of 20|il using 400 units of T4 DNA ligase (NEB). Sticky end ligations w ere incubated at room tem perature for 30m ins-l hour. Blunt end ligations w ere incubated longer, typically overnight, also at room tem perature.
8.2.9 PCR
For generating fragm ents for sub cloning, high fidelity V ent or Pfu polym erases w ere used. Typically a lOOpl reaction w as set up in buffer supplied w ith the enzyme. A standard protocol was used for all fragm ents of IK bp in length or less: 5mins@ 94°C, 30x(l m in at 94°C, 1 min@40°C, 1
min@72°C) and lOmins at 72°C. For longer fragm ents the extension time was increased. W hen screening bacterial colonies, or checking the genotype of yeast strains, TAQ polymerase produced by the LC.R.F. central services was used, in a buffer of lOmM Tris.Cl pH8.3, 50mM KCl, l.SmM MgCl2, 0.1% gelatin.
8.2.10 DNA sequencing
All sequencing was perform ed using either an ABI or an ALF autom ated sequencer, which were operated by the LC.R.F sequencing service.
8.2.11 Annealing single stranded oligonucleotides
Ip g of each oligo dissolved in TE w as added to a m icrofuge tube, and placed in a beaker of boiling water. The w ater was then allowed to cool slowly to room temperature.
8.2.12 End labelling oligonucleotides for EMSA probes
5ng of double stranded oligo was mixed w ith 12pl H2O, 2pl lOx FNK
buffer (700mM Tris.Cl pH7.6, lOOmM M gC h 5mM DTT), 3pl of 32p dATP and 10 units of polynucleotide kinase. The reaction w as incubated at 37°C for 20mins. U nincorporated nucleotides were rem oved using a sephadex G-50 column.
8.2.13 Electrophoresis and blotting of RNA
16|il of DMSO, 3.2 )1 1 phosphate buffer pH6.5, and 5|il of 6.6M deionised
glyoxal w ere added to 10|xl of RNA sam ple containing 10-25jig RNA. The m ixture w as heated to 50°C for ISmins. 8|il of RNA loading dye w as added
(50% glycerol, lOmM phosphate buffer pH6.5, 0.4% brom ophenol blue), and the sam ple subjected to electrophoresis in a 1.2% agarose gel buffered by 1 0-
15mM phosphate buffer ,pH6.5. Gels were run at a m axim um of 4V per cm. The RNA w as blotted onto a 'Gene-Screen' m em brane (NEN R esearch Products) and fixed w ith UV light, using a 'Stratalinker' (Stratagene). The blot was w rapped in cellophane, and stored at -20°C.
8.2.14 Radiolabelling of probes for northern blotting
DN A fragm ents to be labelled w ere generated by PCR or restriction digestion and purified from an agarose gel using a 'Gene Clean 11' (Bio 101 Inc.) or 'Jetsorb' Kit (AMS Biotechnology). 50ng of DNA was labelled w ith ^^P dCTP using a 'M egaprime' Kit (Amersham). U nincorporated nucleotides w ere not rem oved from the labelled probe prior to use. The probe was heated to 100°C for 5mins before adding to the hybridisation tube.
8.2.15 Northern blot hybridisation and washing conditions
Blots were prehybridised for 1-3 hours at 42° C in 20mls of 6x SSC, 5x
denhardts, 5% SDS, 50% formamide, 10% dextran sulphate and lOOpg/ml heat denatured, sonicated salmon sperm DNA. The heat denatured probe was then ad d ed directly to the prehybridisation solution, and hybridisation left to p ro ceed for 14-20 hours at 42°C. Blots w ere w ashed w hile still in the hybridisation tube, using two 45 m in w ashes at 65°C in 2xSSPE/2%SDS, followed by a 30 min w ash at 42°C in 0.2xSSPE/0.5%SDS.