Kit Supplier
Antarctic Phosphatase New England Biolabs
BsmBI New England Biolabs
DpnI NEB / Fermentas
EcoRV New England Biolabs
Gene Ruler DNA Ladder Mix Fermentas Gene Ruler 1 kp DNA-Leiter Fermentas High Pure PCR Cleanup Micro Kit Roche High Pure Plasmid Isolation Kit Roche
Lysozyme Sigma PageRuler Unstained Protein Ladder Fermentas
Phusion DNA Polymerase Finnzyme QIAquick Gel Extraction Kit Qiagen RNA from Bacteriophage MS2 Roche Rapid DNA Ligation Kit Fermentas
T4 Polynukleotidkinase New England Biolabs Vent (exo-) DNA polymerase New England Biolabs 5.3 Bacterial strains and plasmids
Bacterial strain and plasmids Source
electro competent E. coli XL10-Gold Stratagene (kindly provided by N.
Staiger) E. coli BL21 (DE3) Gold Stratagene
pGDR11 AG Welte, University of Konstanz158
pASK-IBA37plus IBA
5.4 Disposables
Disposable Supplier
96-well plates (200 μl) ABgene
96-deep well plates (2.2 ml) Peske
384-deep well plates (300 μl) Abgene
Adhesive sealing foil ABgene
Cuvettes (1.5 – 3 ml) Roth
Diamond sealing foil ABgene
Electroporation Gene Pulser cuvettes (1/2 mm) BioRAD
Gas permeable adhesive seals ABgene
Glass wool Serva
Injection needles Braun
Petri dishes Roth
Reaction tubes (1.5, 2.0 ml) Peske Laborbedarf
Scalpels Bayha
Sephadex G-25 columns GE Healthcare
Tips for pipetting robot Hamilton Robotics
Tips for multichannel pipettes Peske
Tips for laboratory pipettes Peske, Eppendorf
UV-cuvettes Eppendorf
Vivaspin columns (6, 20 ml) 10.000 MWCO Sartorius stedim biotech
Whatmanpaper 3mm Merck Eurolab
5.5 Equipment
Instrument Supplier
Agarose gel racks Fisher Scientific, ABgene
Autoclav Autoclav Systec 3150 ELV
Instrument Supplier
ESI-MS 3000 Bruker Daltonics
Floor centrifuge 5804R Eppendorf;
Freezer (-80°C, -20°C, 4°C) Thermo Forma, Liebherr, Premium Gel documentation device, Chemidoc XRS BioRAD
Gel dryer BioRAD
GenePix Personal 4100A microarray scanner Molecular Devices
Heating block Stuart
Incubation shaker New Brunswick Scientific
Incubation shaker, Titramax 1000 Heidolph
Laboratory pipettes Eppendorf
Magnetic stirrer MR 3000 D Heidolph
Microwave oven Micromaxx
Microarray Peltier Thermal Cycler 200 MJ Research
Multifuge KR 4 Heraeus
Multichannel pipettes Transferpette Brand Nanoplotter 2.0 system Oligonucleotide synthesizer ABI 392
PAGE electrophoresis racks BioRAD
PCR-thermocycler Biometra
pH meter, Seven Easy Mettler Toledo
Phosphorimager Molecular Imager Chemi-Doc XRS System
BioRAD
Photometer Cary 100 Bio Varian
Pipetting robot Microlab Star Hamilton Robotics
Phosphor screens Fuji
Instrument Supplier
Phosphor screen cassettes BAS-Cassette 2025 Fuji
Plate sealer Abgene
Power supply Power Pac 3000 BioRAD Radioactivity counter Contamat FHT111M Thermo Reagent dispenser Multidrop Thermo Real-time PCR-thermocycler:
Chromo4 Lightcycler 480
BioRAD Roche Refrigerated centrifuge Biofuge Primo R Heraeus
Radioactivity shields Roth
SDS-PAGE racks BioRAD
Speed-Vac Concentrator 5301 Eppendorf
Sterile bench HERA safe
Thermomixer, Thermomixer comfort Eppendorf Table top centrifuges, MiniSpin Eppendorf
UV-transilluminator Bachofer
UV/VIS photometer ND-1000 Nanodrop Peqlab
Vortexer REAX Control Heidolph
Water baths Memmert
5.6 Buffers and solutions
Buffers and solutions Components Concentration Agarose gel loading buffer Bromophenol Blue
Xylene cyanol FF 1 x KlenTaq lysis buffer 1 x KlenTaq reaction buffer
Lysozyme 0.1 mg/ml
1x KlenTaq storage buffer Tris-HCl pH 9.2 (NH4)2SO2
SDS-PAGE stacking gel
SOB-Medium pH 7.0 Tryptone Yeast extract
Buffers and solutions Components Concentration Urea-PAGE stock solution 2 Acrylamide solution
Urea
1x Pfu reaction buffer Tris-HCl pH 8.8 MgSO4
5.7 Determination of DNA concentration
Quantification of DNA and RNA was conducted by UV absorption measurements at 260 nm using UV/VIS photometer. Water or the respective buffer was used as a reference.
Based on Lambert-Beer's law the DNA concentration could be calculated. Extinction coefficients, as well as molecular weight and melting behaviour were determined using the online available “Oligonucleotide Properties Calculator”
(http://www.basic.northwestern.edu/biotools/oligocalc.html)159 which is based on specific extinction coefficients of each different nucleobase.
5.8 Oligonucleotides
Oligonucleotides were purchased from Metabion international AG (Martinsried, Germany) or Purimex (Grebenstein, Germany). All chemically modified oligos, oligos for allele specific reactions and kinetic characterisations were purified by denaturing PAGE. Templates and cloning primers were used desalted or 1x HPLC purified.
The integrity of all oligonucleotides was confirmed by ESI-MS. Therefore, oligonucleotide samples were diluted (c = 1μM) in a mixture of water (79% v/v), 2-propanol (20% v/v) and triethylamine (1% v/v).
5.9 Radioactive labelling of DNA-oligonucleotides
Oligonucleotides were radioactively labelled by [32P]-ATP using T4 polynucleotide kinase (T4-PNK). The labelling reaction was carried out in 50 μl scale with 400 nM oligonucleotides, 20 μCi [32P]-ATP (2 μl), 0.4 U / μl T4-PNK (2 μl) and 1x T4-PNK reaction buffer. The reaction was incubated at 37°C for ~1 h and subsequently stopped by heat-inactivation of T4-PNK at 95°C for ~5 min. After gel filtration (G25 sephadex spin column) to remove salt and residual [32P]-ATP, the radioactively labelled oligonucleotide solution was diluted with 19 μl of a 10 μM unlabelled oligonucleotide . applied RT temin a final primer concentration of 3 μM.
5.10 Agarose gelelectrophoresis
Agarose gel electrophoresis was performed according to standard procedures.
Depending on fragment DNA size a 0.8% or 2.5% agarose in 0.5 x TBE was used. Gels were stained for 5-30 min in ~0.01% (w/v) ethidiumbromide (EtBr) in 0.5 x TBE and shortly destained in 0.5 x TBE for further 5 min and documented in a Chemidoc XRS System. For preparative agarose-gels it was taken care that UV irradiation of the fragments to be isolated was minimised.
5.11 Denaturing polyacrylamide gelelectrophoresis
Denaturing polyacrylamide gel electrophoresis (PAGE) was performed according to
contained urea. Preparative gels had a gauge of 1.5 mm and analytical gels a gauge of 0.4 mm.
After running, analytical gels were transferred onto whatman paper, dried subsequently and exposed to a phosphor screen preferentially overnight.
For DNA-isolation from polyacrylamide-gels the separated DNA was visualised by UV-shadowing and the respective bands were excised with a scalpel. Excised gel pieces were crushed by forcing them through a syringe and collected in 2 ml eppendorf tubes.
DNA was eluted from crushed gel pieces by adding H2O and incubation at 55 °C overnight. Next, the DNA/PAGE mixture was filtrated through silanised glass-fibres wool. The filtrate was dried in a speedvac (Eppendorf) and extracted DNA was purified by ethanol precipitation according to standard procedures.
5.12 SDS polyacrylamide gelelectrophoresis
Expressed proteins were analysed by discontinuous glycine SDS-PAGE (12%) according to standard procedures including a stacking- and a resolving-gel. Staining was performed by a Coomassie-staining solution.
5.13 Determination of protein concentration
Protein samples were loaded in parallel with a BSA standard dilution series. Respective band intensities were quantified on a BioRad Chemidoc XRS System (Quantity One 4.5.0) and protein concentrations could be calculated in comparison with the BSA sample intensities by linear curve analysis.
5.14 Site directed mutagenesis
In general site directed mutagenesis was performed according to the QuikChange Site-Directed Mutagenesis protocol (Stratagene) with the following modifications: Desalted mutagenesis primers and Phusion DNA Polymerase was used for whole plasmid PCR.
Negative controls were performed via plating DpnI digested original plasmid on LB-agar plates with 100 ng/μl carbencillin.
5.15 Transformation of chemically competent cells
Chemically competent E. coli cells were transformed as described in the manufacturer’s manual (Stratagene) using an electroporator Gene Pulser Xcell.
5.16 DNA-sequencing
The prepared DNA samples were sent to a professional sequencing company (GATC Biotech AG, Konstanz). Sequences were analysed and aligned using Chromas Lite (http://www.technelysium.com.au/chromas_lite.html) and SDSC Biology Workbench (http://workbench.sdsc.edu/).
5.17 Crystal structure models
Own DNA and protein crystal structure models were prepared by the respective PDB-code using PyMOL Molecular Viewer ( http://pymol.sourceforge.net/ ) for visualisation.
5.18 Methods for section 2.1
5.18.1 Real-time PCR experiments
Real-time PCR was performed as described by using an iCycler system (BIORAD). In brief, the reactions were performed in an overall volume of 20 μL containing 400 pmol of the respective templates, in the appropriate buffer provided by the supplier for Vent (exo-) DNA polymerase (20 mM tris(hydroxymethyl)aminomethane-HCl (Tris-HCl; pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%Triton-X100). The final mixtures contained dNTPs (200 μM each of dATP, dGTP, dCTP and TTP), primers (0.5 μM each of the respective primer probe and reverse primer), 0.4 units Vent (exo-) DNA polymerase (New England Biolabs; units defined by the supplier) and a 1/50 000 aqueous dilution of a SybrGreen I 10,000 solution in dimethylsulfoxide (DMSO). All PCR amplifications
for 35 s and extension at 72°C for 40 s. The presented results are from at least three repeated independent measurements of duplicates that originated from one master mix. Identical template-target, primer-probe and reverse-primer DNA sequences were used to those employed in earlier studies for comparison24.
5.18.2 Primers and templates
Oligonucleotide Sequences are in the Farber disease109 context.
primer probes (20nt)
5’-d(CGTTGGTCCTGAAGGAGGAT*)with T*: T, 2ST, 4ST, TOMe, 2STOMe or 4STOMe. reverse primer (20nt)
5’-d(CGCGCAGCACGCGCCGCCGT) target template (90nt)
5’-d(CCGTCAGCTGTGCCG TCGCGCAGCACGCGCCGCCG TGGACAGAGGACTGCAGAAAATC AACCT N TCCTCCTTCA GGACCAACGTACAGAG) with N= A or G.
template (33nt)
5’-d(AAATCAACCT N TCCTCCTTCAGGACCAACGTAC)-3’ with N= A or G.
5.18.3 DNA thermal-denaturation studies
Melting curves were recorded on a Cary 100 bio UV/Vis instrument with temperature controller. Data were obtained from three individual cooling/heating cycles. Melting temperatures (Tm values in °C) were obtained from the maximum of the first derivative of the melting curves (absorbance at 260 nm versus temperature). Measurements were conducted in the appropriate Vent (exo-) DNA polymerase buffer (ThermoPol, New England BioLabs) without Triton-X100 (20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4) and contained 4.75 μM duplex DNA. The mixtures were heated at 95°C for 5 min and slowly cooled down to room temperature prior to the melting curve measurements. A measurement of the buffer was conducted separately and subtracted from the spectra resulting from the sample. Shorter templates (33nt, A/G) and respective primer probes containing T, 2ST, 4ST, TOMe, 2STOMe or 4STOMe as above were used.
5.18.4 Circular dichroism spectra
CD spectra were recorded on a Jasco 720 instrument in ThermoPol buffer (New England Biolabs; 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1%
Triton-X100) at room temperature. The samples contained 4.75 μM duplex DNA. All mixtures were heated to 95°C for 5 min and allowed to cool slowly to room temperature prior to measurements. A spectrum of the buffer was measured separately and subtracted from the spectra resulting from the samples. An average of 12 spectra was recorded in each experiment. The sequences were as described in the previous Section 5.18.3.
5.18.5 Kinetic single-nucleotide incorporation studies
Single-nucleotide incorporation reactions contained varying enzyme amounts (0.8–8 nM) and primer (150 nM)–template (300 nM) complex in ThermoPol buffer. Primer–
template complexes were annealed and the reaction was initiated by addition of different concentrations of dATP in solution. After incubation for different times at 72°C, reactions were quenched by addition of two reaction volumes of gel-loading buffer (80% formamide (v/v), 20% water (v/v), 20 mM ethylenediamine tetraacetate (EDTA)) and the product mixtures were analysed by 12% denaturating PAGE.
Incorporation quantities were measured by quantifying the intensity of each band produced by the DNA polymerase by using a Phosphorimager. From this quantification, the amount of incorporated nucleotide was calculated. The intensity of the background was subtracted from each band. The reaction conditions were adjusted for different reactions to allow 20% or less of primer extension, thereby ensuring single-completed-hit conditions according to published procedures111. Steady-state KM and kcat values were obtained by fitting with the Hanes–Woolf equation. The presented results are from measurements that were repeated independently at least three times.
5.19 Methods for section 2.2
5.19.1 Primers and templates
The integrity of all primer probes was evaluated by ESI-MS.
primer (23nt)
5’-d(GAC CCA CTC CAT CGA GAT TTC TC)-3’
single stranded template (35nt)
5´-d(GCG CTG GCA CGG GAG AAA TCT CGA TGG AGT GGG TC)-3´.
Farber primer T/C (20nt)
5´-NH2(CH2)6-d(CGT TGG TCC TGA AGG AGG A T/C)-3´
Leiden primer T/C (25nt)
5´-NH2(CH2)6-d(CAA GGA CAA AAT ACC TGT ATT CCT T/C)-3´
DPyD primer T/C (25nt)
5´-NH2(CH2)6-d(GTT TTA GAT GTT AAA TCA CAC TTA T/C)-3´
Farber templates A/G (90nt)
5’-d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT A/G TC CTC CTT CAG GAC CAA CGT ACA GAG)-3’
Leiden templates A/G (98nt)
5’-d(GAC ATC ATG AGA GAC ATC GCC TCT GGG CTA ATA GGA CTA CTT CTA ATC TGT AAG AGC AGA TCC CTG GAC AGG C A/G A GGA ATA CAG GTA TTT TGT CCT TG)-3’,
DPyD templates A/G (120nt)
5’-d(AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA AAA CGG CTG CAT ATT GGT GTC AAA GTG TCA CTG AAC TAA AGG CTG ACT TTC CAG ACA AC A/G TAA GTG TGA TTT AAC ATC TAA AAC)-3’.
5.19.2 Activation of glass slides and spotting of amino-modified oligonucleotides to glass slides
Aminopropyl-silylated glass slides were derivatized with 1,4-phenylene diisothiocyanate (0.2% (w/v)) in a pyridine/dimethylformamide (10% (v/v)) solution for 2h at room temperature. The slides were subsequently washed several times with dimethylformamide and acetone, dried under a stream of nitrogen and stored desiccated until spotting. Spotting of 5’-amino modified primer probes (20 μM), ~4 nl per spot, in sodium phosphate buffer (150 mM, pH 8.5), was performed between 19-22°C and 70-77% humidity. The slide tray was cooled during spotting procedure at 10°C. After the spotting process the slides were incubated at room temperature in a closed petri dish over a saturated NaCl solution overnight. Thereupon, blocking was performed in a NH4OH solution (10%) for 30 min, followed by subsequent washing steps with water. The slides were dried under a stream of nitrogen and stored at 4°C until further use.
5.19.3 Primer extension and arrayed primer extension
Wild-type and mutant Pfu DNA polymerase was obtained as described72.
The reactions for the primer extension reactions in solution contained 1x Pfu reaction, dATP, dGTP, dCTP (each 200 μM), single stranded template (200 nM), 5´-[32P]-labelled primer (150 nM) and Pfu DNA polymerase (100 nM) within an overall volume of 20 μl.
TTP and F3-dUTP concentration was varied (200 μM TTP, 185 μM TTP + 15 μM F3-dUTP (7.5%), 200 μM F3-dUTP). The reaction mixtures were denatured for 2 min at 95°C, annealed at 55°C and the reaction was initiated by addition of DNA polymerase (100 nM). After 20 min at 72°C, the primer extension was stopped by addition of gel-loading buffer. Product mixtures were separated by denaturing PAGE (12%). Gels were analysed with a Molecular Imager by phosphor imaging and via Cy3-fluorescence detection channel.
The arrayed primer elongation reactions contained 1x Pfu reaction buffer, dATP, dGTP, dCTP (200 μM), TTP (185 μM), F3-dUTP (15 μM), single stranded template (500 nM),
with round cover slips (10 mm diameter). Primer extension was carried out with a microarray thermocycler using the following temperature steps: 95°C for 225 sec., 55°C for 60 sec. and 72°C for 20 min. The reaction was stopped by cooling the slides to 4°C and the slides were subsequently washed under gentle agitation in 0.1 x SSC buffer (sodium chloride / sodium citrate) + SDS (0.1%) twice for 10 min and water three times 5 min. The slides were then dried under a stream of nitrogen directly before reading out with a GenePix microarray scanner machine.
5.20 Methods for section 2.3
5.20.1 Primers and templates on solid support abasic site primer-template
p 1 5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-3´
t 1 5´- AAAACGC TGTAGCA TAG AGT ACATG ACA G TT CX CCT A TC CTC CTT CAG GAC CAA CG-3´ X = stable abasic site analogue
fully matched primer-template
p2 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2 5´-AAA GCT CCT TTC TGA ATA TTG AGC TCA TCA GTG AGA AAA CGG CTG CAT ATT GGT GTC AAA GTG TCA CTG AAC TAA AGG CTG ACT TTC CAG ACA AC A TAA GTG TGA TTT AAC ATC TAA AAC-3´
single mismatched primer-template
p3 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2 (sequence see above)
distal mismatched primer-template
p4 5´- NH2C6-GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2 (sequence see above)
triple mismatched primer-template
p5 5´- NH2C6-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2 (sequence see above)
surface coverage estimation
p6 5´- NH2C6-CGT TGG TCC TGA AGG AGG AT-biotin-3´
5.20.2 Primers and templates in solution experiments abasic site primer-template
p1´ 5´-CGT TGG TCC TGA AGG AGG AT-3´
t1´ 5´-AAA TCA XCC TAT CCT CCT TCA GGA CCA ACG TAC-3´
X = stable abasic site analogue
fully matched primer-template
p2´ 5´-GTT TTA GAT GTT AAA TCA CAC TTA T-3´
t2´ 5´-CTT TCC AGA CAA CAT AAG TGT GAT TTA ACA TCT AAA AC-3´
single mismatched primer-template
p3´ 5´-GTT TTA GAT GTT AAA TCA CAC TTA C-3´
t2´ (sequence see above)
distal mismatched primer-template
p4´ 5´- GTT TTA GAT GTT AAA TCA CAC CTA T-3´
t2´ (sequence see above)
triple mismatched primer-template
p5´ 5´-GTT TTA GAT GTT AAA TCA CA GAA AT-3´
t2´ (sequence see above)
5.20.3 Overexpression of KlenTaq clones in multiwell format and cell lysate preparation
KlenTaq clones were parallel expressed in 96x deep-well plates containing 1 ml of LB-medium and 100 μg/ml carbenicillin at 37°C. Overexpression was induced via addition of 200 μg/l anhydrotetracycline at OD600~0.8 for 4h. Afterwards, the plates were centrifuged at 4000 x g for 15 min, the supernatants were thrown away and cell pellets were resuspended in 1x KlenTaq reaction buffer containing an additional amount of lysozyme (0.1 mg/ml). The sealed plates were subsequently incubated for 15 min at 37°C and then for 40 min at 75°C. After centrifugation at 4000 x g for 45 min the lysate was directly used for screening.
5.20.4 Spotting of DNA polymerase cell lysate mixtures and screening reactions Immediatly before spotting, the DNA polymerase cell lysates were mixed 1:1 with a KlenTaq reaction mix containing 1xKlenTaq buffer, 0.2 % BSA, 400 μM dATP,dGTP,dCTP, 380 μM dTTP, 20 μM Biotin-11-dUTP and the respective templates in a 96x well plate at 4°C and agitated for 30 sec using a shaker module. The cell lysate mastermix mixtures were centrifuged for 1 min at 4000 x g. Spotting was then performed at ~20°C air temperature and air humidity between 60-70%. The slide tray was also cooled during the whole spotting procedure at 5°C. Cell lysates were spotted in a way that each lysate covered 2 columns and all 5 different primer spot rows with 3 drops per spot resulting in approx. 1.2 nl total spotting volume. The slides were subsequently placed in a humidity chamber consisting of a water filled petri dish at room temperature for ~1 minute until all spots were rehydrated. After incubation, the slides were shortly rinsed twice under gentle agitation in 0.1 x SSC+SDS buffer (15 mM NaCl, 15 mM sodium citrate, pH 7.0, 0.01% SDS) for 5 min and twice with water (5 min each). The slides were then dried under a stream of nitrogen. Afterwards, 70 μl of a streptavidine-alexa fluor® 546 solution 0.4 μg/ml in 1x TBS-T buffer (10 mM Tris-HCl pH 8.0 150 mM NaCl, 0.05% Tween-20) was placed on each slide and directly covered with a square glass cover slips 24 x 60 mm. Incubation was carried out in a humidity chamber (see Figure 21) at room temperature for another 40 min. The cover slip was then removed and the slides were rinsed twice with 1x TBS-T buffer and once with
water. Afterwards, the slides were dried under a stream of nitrogen before readout with a GenePix microarray scanner machine (532 nm channel, 10 μm / pixel resolution) was performed.
5.20.5 Expression and purification of KlenTaq mutants
KlenTaq wt and mutants were purified in batch technique using Ni-NTA sepharose. In detail 100 ml cultures were grown to an OD600 of 1.0 at 37°C following induction with AHT (200 μ g/L) for 3 h. After harvesting cells were lysed for 15 min at 37°C in 5 ml KlenTaq lysis buffer followed by heat denaturation of host proteins at 75°C for 40 min and centrifugation at 20.000 x g for 30 min. Supernatants were incubated with Ni-NTA sepharose slurry. After washing with KlenTaq washing buffer (5 mM and 20 mM imidazol), elution was carried out using washing buffer (200 mM imidazol). After buffer exchange to 2x KlenTaq washing buffer, Tween20, (NH4)2SO4 and glycerol was added to 50 % (see Buffers and solutions, KlenTaq storage buffer). Enzyme purity (Figure 27) and quantity was controlled and determined by SDS-PAGE using an albumin standard dilution curve. Purified enzymes were stored at -20°C.
5.20.6 Primer extension reactions in solution
20 μl of the reaction contained 1x KlenTaq buffer, 100 μM dNTPs, 225 nM template, 150 nM 5´-32P-labeled primer. DNA polymerase concentrations were 1 nM for matched, mismatched and distal mismatched cases and 50 nM for triple-mismatched and abasic site templates. The reaction mixtures without dNTPs were annealed by heating for 2 min at 95°C and cooling to 25°C. Reactions were initiated by addition of dNTPs. After incubation at 51°C for 15 min in cases of match, mismatch and distal mismatch and 1h for the triple-mismatch and abasic site containing reactions, the primer extension was stopped by addition of 40 μl of gel-loading buffer (80%
formamide, 20 mM EDTA). Product mixtures were separated by 12% denaturing PAGE and analysed by phosphorimaging.
5.20.7 Test of the influence of drying and rehydration on the KlenTaq polymerase activity in solution
Solutions of KlenTaq wt ranging from 400, 40, 4, 0.4, 0.04, 0.004 nM in 1x KlenTaq reaction buffer were divided in two 10 μl aliquots, respectively. One aliquot was dried under vacuum using a high-vacuum membrane pump while the other aliquot was stored at room temperature. After 1 h, the dried residue was dissolved in 10 μl water and both aliquots were used for subsequent primer extension reactions using fully matched primer p2´ and template t2´. Reactions (20 μl) contained 1 x KlenTaq buffer, 100 μM dNTPs, 225 nM template, 150 nM 5´-[32P]-labelled primer and 2 μl of the DNA polymerase containing solution. The reactions were stopped after 15 min incubation at 51°C by addition of 40 μl of gel-loading buffer. The products were separated by 12%
denaturing PAGE and analysed by phosphorimaging (see Figure 22).
5.20.8 Spotting and immobilisation of short DNA oligomers
Activation of glass slides and spotting of amino-modified oligonucleotides to glass slides were conducted as previously described (see Section 5.19.2)
5.20.9 Estimation of surface coverage of oligonucleotides bound to the chip surface
To estimate the loading density and the amount of attached DNA primers on the
To estimate the loading density and the amount of attached DNA primers on the