Resultados Generales Pretest y Postest

CL-6B sepharose 0.5ml centrifuge tube 425-600 micron glass beads

- Hole punched in base

1.5ml centrifuge tube

15ml tube

Eluate

Figure 2.2: Set-up of CL-6B sepharose column for purifying annealed DNA.

C h apter 2: M aterials an d M ethods

ddNTP’s, prewarmed to 37°C and incubated at 37°C for 5min. 4pl o f stop solution (95% w/v formamide, 20mM EDTA pH8.0, 0.05% w/v bromophenol blue, 0.05% w/v xylene cyanol) was then added and the samples stored at -20°C until needed.

2.1.13 Analysis of sequencing reactions by 7M polyacrylamide urea gel electrophoresis

Sequencing reactions from Section 2.1.12 were analysed on a 7M urea polyacrylamide wedged sequencing gel. 75g of molecular biology grade urea (Sigma) was dissolved in 30ml of 40% bis-acrylamide (Scotlab), 15ml o f filtered TBE (see Section 2.1.3) and 45ml of SDW in a plastic jug covered with cling film on a heated stirrer at 50°C for 1 hour or until the urea had completely dissolved. During this time, the gel plate assembly was set up. Briefly, two 33 x 43cm and 5mm thick glass plates (Gibco) were thoroughly cleaned with 70% ethanol and the shorter plate then siliconised in a fume hood with dichlorodimethylsilane (Sigma). Two spacers (430 X 0.4mm) were positioned at the outer edges of the larger plate to separate the

plates and two additional 20 x 0.4mm spacers placed at the bottom. The shorter plate was then placed on top and the plates and spacers tightly taped together. When the gel had cooled to 40°C, 900pl o f 10% w/v ammonium persulphate (APS) (Sigma) and 80pl o f N,N,N’,N’ tetramethylethylenediamine (TEMED) (Sigma) were added and mixed well. This was poured into the assembled gel plates. Two 24 well shark-tooth combs (Sigma) were then placed between the gel plates with their flat side against the poured gel before it set, this ensured the exposed edge o f the gel was flat. The gel was allowed to set at room temperature for 30 minutes, and then placed in a gel tank. The combs were carefully removed and turned around to form sample wells and any unpolymersised acrylamide or residual urea was removed with Ix TBE and a fine tipped pippette. The gel was pre-warmed by running in Ix TBE at 40V for 30min.

Samples were heated at 94°C for 2min and 5pi of each sample (Section 2.1.12) was loaded in the order ‘G ’, ‘A ’, ‘T ’, ‘C’. Samples were electrophoresed at 75W until the bromophenol blue dye was about 3cm from the bottom of the gel (a long run). At this point the same samples were loaded again into new wells and the gel run again until the bromophenol blue dye was 3cm from the bottom o f the well (a short run). The gel

C h apter 2: M aterials a n d M ethods

plates were separated leaving the gel attached to the non-siliconised plate. The gel was fixed in 10% glacial acetic acid (BDH) for 30min. The gel was blotted with IsT3 cartridge paper (Whatman) and subsequently transferred onto it before being vacuum dried at 80°C for 2.5 hours using a Bio-rad dryer and Aquavac pump. The dried gel was exposed to Hyperfilm MP X-ray film (Amersham) for between 24h and 1 week depending on the activity date o f the [a-^^S]dATP used in the labelling reaction. The DNA sequence was read from the autoradiograph on a light box.

2.1.14 Large scale preparation of plasmid DNA

An endo-free Maxi prep kit (Qiagen) was used to extract DNA on a larger scale from two recombinant pGEM clones; one containing the 233bp wild type 5’UTR insert the other the mutagenised internal standard insert. Briefly, -70°C frozen stocks of the desired clones were streaked out onto agar plates supplemented with ampicillin and X-gal (see Section 2.1.10) and incubated overnight at 37°C. Single colonies were then picked, 5ml starter cultures set up from each in LB broth supplemented with 50pg/ml ampicillin and incubated overnight at 37°C in a shaking incubator (~250rpm). This culture was diluted 1/500 (200pl) into 100ml o f LB containing 50|ig/ml ampicillin and grown at 37°C for 16 hours in a shaking incubator (~250rpm). The cells were harvested at 6000ipm for 15min at 4°C in an IECPR-7000 centrifuge and the bacterial pellets completely resuspended in 10ml o f PI cell resuspension buffer (50mM Tris-HCl pH 8.0, lOmM EDTA, lOOpg/ml Rnase A). 10ml of P2 cell lysis buffer (200mM NaOH, 1% SDS) was then added, and the lysate mixed by inverting 4-6 times, before incubation at room temperature for 5min. 10ml of chilled P3 neutralisation buffer (3.0M potassium acetate pH 5.5) was added, the lysate inverted 4-6 times to mix, immediately poured into the barrel of a stoppered Qiafilter cartridge and incubated at room temperature for lOmin. The cap was removed from the outlet nozzle and the cell lysate filtered into a 50ml tube using a plunger. 2.5ml of buffer ER (composition withheld by supplier) was added to the filtered lysate mix by inverting ~10 times and incubated on ice for 30min. Meanwhile, a Qiagen-tip 500 was equilibrated by adding 10ml of QBT equilibration buffer (750mM NaCl, 50mM MOPS pH7.0, 15% isopropanol, 0.15% Triton X-100) and allowing the column to empty by gravity flow. The filtered lysate was placed on

C h apter 2: M aterials a n d M ethods

the column and allowed to enter the resin by gravity flow. The flowthrough was discarded and the column washed twice with two 30ml aliquots of QC wash buffer (l.OM NaCl, 50mM MOPS pH 7.0, 15% isopropanol). The DNA was eluted with 15ml of QN elution buffer (1.6M NaCl, 50mM MOPS pH 7.0, 15% isopropanol), precipitated with 10.5ml (0.7vol) of room temperature isopropanol and centrifuged immediately at 24000rpm for 30min at 4°C in a Beckman L-80 ultracentrifuge. The supernatant was decanted off and the pellet washed with two 2.5ml aliquots of endotoxin free room temperature 70% ethanol and air dried for 20min. The pellet was resuspended in lOOpl o f endotoxin free SDW. The concentration o f the DNA was determined by optical density at 260nm in a Genequant II RNA/DNA calculator (Pharmacia Biotech) where an OD260 of 1.0 is equivalent to 50pg o f double stranded DNA. The stock DNA was stored at -20°C until needed.

2.1.15 Linearisation of plasmid prior to transcription

Recombinant pGEM wt and control vectors were initially linearised with Spe I prior to the transcription reaction to produce positive sense RNA o f a discrete length (287nt). 4pg of each o f the plasmids was digested with 1 pi Spe I (lOu/pl Promega) in 2pl of lOx Buffer B (60mM Tris-HCl, 60mM MgCb, 500mM NaCl, lOmM DTT pH 7.5) for 3h. 0.5 pi o f the uncut and linearised plasmid was added to 5 pi o f loading dye and run on a 0.8% agarose gel with 1 pi o f high molecular weight Eco R l / Hind III X- DNA markers (Promega) at lOOV and 130mA for 55min to verify complete linearisation of the two vectors.

2.1.16 In-vitro transcription using T7 RNA polymerase

The linearised vectors were used for in vitro transcription using the RiboMAX™ large scale RNA production system (Promega) to make positive sense RNA transcripts. 750ng o f linearised pGEM vector DNA was transcribed in the following reaction mixture: 20pl 5x transcription buffer (400mM HEPES-KOH pH7.5, 120mM MgCl], lOmM spermidine, 200mM DTT), 30pl rNTPs (25mM each o f GTP, ATP, UTP and CTP), lOpl o f T7 enzyme mix (300u/pl T7 RNA polymerase, 15u/plRNasin

C h apter 2: M aterials a n d M ethods

ribonuclease inhibitor, 190u/ml yeast inorganic pyrophosphate) made up to lOOpl with nuclease free water. The reaction was incubated in a water bath at 37°C for 3h and frozen at -70°C until needed.

2.1.17 Removal of DNA template with RNase free DNase 1

The pGEM DNA template was removed from the transcription reaction by digestion with RNase free DNase 1 (Ambion). 50pl of transcription reaction was treated with 2pl of DNase 1 (2u/pl) made up to lOOpl with nuclease free water and incubated for 15min at 37°C.

2.1.18 Clean up of RNA

The resultant RNA preparation was cleaned up using an RNeasy kit (Qiagen). The composition o f certain buffers in this procedure cannot be given here, as they are not revealed by the manufacturer. Briefly, 350pl of RLT lysis buffer (lOpl of p- mercaptoethanol added to 1ml of RLT buffer before use) was added to lOOpl of DNase treated RNA (Section 2.1.18) followed by 250pl o f 100% ethanol, mixed by pippeting and loaded onto an RNeasy spin column inserted into a 2ml centrifuge tube. The column was then spun at lOOOOrpm for 15sec in a Jouan M 1 4 .il bench top centrifuge. The flow through was discarded, the column washed with SOOpl of RPE column wash buffer (4vol o f ethanol added before use) and centrifuged at lOOOOrpm for 15sec. The wash step was repeated with the spin increased to 2min to dry the membrane. The RNA was finally eluted from the colunm with 50pl o f nuclease free water.

2.1.19 MOPS/ formaldehyde gel electrophoresis of RNA transcripts

5|xl of purified DNase treated RNA was analysed on a 1% MOPS formaldehyde agarose gel along with RNA markers (Promega). Ig of agarose (Bioline) was added to 85.2ml DEPC (diethylpyrocarbonate) treated water (Sigma) and 10ml of lOx