III. PROCEDIMIENTOS METODOLÓGICOS
3.8. Selección de documentos
2 .1 . Materials 2 .1 .1 . Chemicals
Adenine Hemisulphate Agarose
Alpha satellite chromosome 7 centromere probe Ammonium Acetate Ammonium Persulphate Bactoagar Bacto-tryptone Bacto-yeast extract Boric Acid
Bovine Semm Albumin (BSA) Bromodeoxyuridine Bromophenol Blue Casamino Acids Chloroform Citifluor Colchicine Cot-1 DNA 4,6-diamidino-2-phenylindole (DAPI) Decon
Deoxyadenosine 5'[alpha^^P] Triphosphate Deoxycytidine 5'-[alpha^^P] Triphosphate 2 -Deoxyadenosine 5 -Triphosphate 2'-Deoxycytidine 5 -Triphosphate 2'-Deoxyguanosine 5'-Triphosphate 2'-Deoxythymidine 5 -Triphosphate Dextran Sulphate
Dimethyl Sulphoxide (DMSO) DNA Ladder (lOObp)
DNA Ladder (Ikb) DNA Ladder (50-500bp) Ethanol
Ethidium Bromide
Ethylenediaminetetraacetic acid (EDTA)
Sigma Gibco BRL Oncor
Sigma Chemical Company Bethesda Research Laboratory Difco Laboratories
Difco Laboratories Difco Laboratories
Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Difco Laboratories
British Drug Houses
UKC Chem Lab, Canterbury Gibco BRL
Gibco BRL Serva
Decon Laboratories Limited Amersham International Amersham International Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim Sigma Chemical Company Fisons Laboratory Supplies Gibco BRL
Gibco BRL Pharmacia
British Drug Houses Sigma Chemical Company Sigma Chemical Company
Fetal Calf Serum (PCS) Formamide
Glacial Acetic Acid Glucose
L-Glutamine Glycerol Hepes
Human Genomic DNA Hydrochloric acid
Hydrolink Long Ranger Gel Isoamyl Alcohol Isopropanol Magnesium Chloride 2-mercaptoethanol Methanol Mineral Oil MOPS
Mutation Detection Enhancement Gel Nonidet P40 Oligonucleotides pd(N)ô Phenol Phytohaemagglutinin Potassium Acetate Potassium Chloride Preservative Free Heparin Saline Sodium Citrate (SSC) Sephadex G-50
Sigmacote Sodium Acetate Sodium Chloride
Sodium Dodecyl Sulphate (SDS) Sodium Hydroxide
Sodium Phosphate
Sonicated Salmon Sperm DNA Spermidine
NNN'N'- Tetramethylethylenediamine (TEMED)
Gibco Life Technologies British Drug Houses British Drug Houses British Drug Houses Gibco BRL
Fisons Laboratory Supplies Sigma
Promega
British Drug Houses J.T.Baker Inc.
Fisons Laboratory Supplies British Drug Houses
Sigma Chemical Company Sigma Chemical Company British Drug Houses Sigma Chemical Company British Drug Houses FMC BioProducts
Sigma Chemical Company Pharmacia
Rathbum Chemicals Gibco BRL
British Drug Houses Sigma Chemical Company Sigma Chemical Company British Drug Houses Pharmacia Fine Chemicals Sigma Chemical Company Fisons Laboratory Supplies Fisons Laboratory Supplies Sigma Chemical Company British Drug Houses British Drug Houses Promega
Sigma Chemical Company LKB Biochrom
Thymidine Tris Base Tris EDTA Tris HCl Triton L-Tryptophan tRNA (E.coli)
Tween (polyoxyethylenesorbitan monolaurate) Tyrosine
Ultra-pure Sequagel, concentrate and diluent Urea
Yeast Nitrogen Base
Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Boehringer Mannheim Sigma Chemical Company Sigma Chemical Company
National Diagnostics, Atlanta, USA Sigma Chemical Company
Difco Laboratories
2. 1. 2. Antibiotics
Ampicillin Kanamycin Gentamicin
(Final conc. 75|ig/ml) (Final conc. 20|ig/ml) (Final conc. 100|ig/ml)
Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company
2. 1.3. Enzymes/B offers Hinc II Hinc II Buffer Klenow Polymerase Proteinase K RNase A Taq Polymerase
Taq Polymerase Buffer Bioxact Polymerase Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim Promega Boehringer Mannheim Bioline Inc. Bioline Inc. Bioline Inc. 2 .1 . 4. Antibodies Fluorescein Avidin DCS Biotinylated Anti-avidin D Anti-digoxigenin-rhodamine
Anti-CD45 (common leucocyte antigen)
Vector Laboratories Vector Laboratories Boehringer Mannheim Boehringer Mannheim
Control antibody for FACS Boehringer Mannheim
2. 1. 5. Solutions (Ready Made at Source)
FACS™CellFix™ FACS™Cell Wash™ FACS™Lysing Solution™ R P M I1640 PBS Polymorphprep™ Becton Dickinson Becton Dickinson Becton Dickinson Gibco BRL Gibco BRL Nycomed
2.1. 6. Solutions (Made On Site)
Cell Lysis Buffer 0.075M NaCl, 0.024M EDTA,
3% SDS, 0.5mg/ml Proteinase K.
Church Buffer 500mls IM sodium phosphate
pH7.2, 350mls 20% SDS, 2ml 0.5M EDTA. Make up to 1 litre with distilled water.
Denaturing Acrylamide Gel (For Radioactive Sequencing)
66ml Ultra-pure Sequagel Diluent, 24ml Ultra-pure
Sequagel Concentrate , 10ml lOx TBE, lOOql Temed, 125^il 10% ammonium persulphate.
Denaturing Polyacrylamide Gel (for Automated Sequencing)
6 ml hydrolink long ranger gel, 21g urea, 6 ml lOx TBE, 12|il Temed, 125|il 10% ammonium persulphate made up to 50 ml with distilled water.
Electrophoresis Loading Buffer lOmM EDTA,
25% w/v bromophenol blue, 50% w/v glycerol
GDIS 1ml Triton, 5mls 10% SDS,
0.5ml Tris HCl, lm l0.5M
EDTA, 1ml 5M NaCl made up to 50ml with distilled water.
Hybridization Mix for FISH Ig dextran sulphate, 4.9mls 2x
SSC, 5mls formamide, 0.1 mis Tween 20.
LB (Luria-Bertani) Medium 5g Bacto-tryptone, 2.5g Bacto-
yeast extract, 5g NaCl, distilled water to 500ml volume. pH adjusted to 7.5 with NaOH.
LB Agar Dissolve 7.5g Bacto-agar in
500ml LB medium.
Maxi Buffer 1.5 Ig Tris HCl, 4.5g glucose,
1.86g EDTA, made up to 500ml with distilled water. pH adjusted to 8.0.
Neutralising Solution 1.5M NaCl, 0.5M Tris-HCl
pH7.5, O.OOIM EDTA
Non-denaturing Polyacrylamide Gel (For SSCP)
25ml mutation detection enhancement (MDE) gel, 69ml deionized water, 6ml lOx TBE, 0.4ml 10% ammonium
persulphate, 0.04ml Temed (total volume 100ml and final MDE gel conentration of xO.5).
lOx Tris-Borate-EDTA (TBE) 108g Tris base, 55g Boric acid, 40ml 0.5M EDTA. Make up to
1 litre with distilled water.
lOx OLB (Probe labelling) Mix solutions A:B:C in the ratio
2:5:3:
Solution A/ml: 625(il 2M Tris, pH8.0, 125|il MgCl2, 25^11 20mM dATP, 25^il 20mM dTTP, 25^1 20mM dGTP, 18|il 2-mercaptoethanol, 157|il water. Solution B: 2M Hepes, pH6.0 with NaOH.
Solution C: pd(N)6
oligonucleotides at OD/ml=90.
0.075M Potassium Chloride 2.794g potassum chloride in
500ml distilled water.
3/5M Potassium Acetate 60ml 5M potassium acetate,
11.5ml glacial acetic acid, made up to 100ml with distilled water.
IM Sodium Phosphate pH7.2 684ml IM Na2HP0 4, 316ml
NaH2P0 4. Make up to 1 litre with distilled water.
1% SDS/0.2M NaOH 5ml 20% SDS, 10ml 2M NaOH,
made up to 100ml with distilled water.
4ST 100ml 20x SSC, 0.25ml Tween
20, made up to 500ml with distilled water.
TE Buffer lOmM Tris-HCl, ImM EDTA, adjust to pH8.0 with conc. HCl.
YAC Broth A: 28g casamino acids, 1 lOmg
tyrosine, 40g glucose, 200mg adenine hemisulphate, made up to
1860mls with distilled water. B: 6.7g yeast nitrogen base made up to lOOmls with distilled water. C: Tryptophan lOOmg in lOOmIs distilled water.
Add A:B:C in ratio 50:5:1
YAC Agar Dissolve 6g Bactoagar in 4G0mls
YAC broth. Add B and C in ratio A:B:C - 50:5:1.
2 .1 . 7. Commercially Available Kits
2 .1 .7 . a). Qiagen Columns (Qiagen)
1. QIAGEN-tip 20 tips 2. Buffer PI 3. Buffer P2 4. Buffer P3 5. Buffer QBT 6. Buffer QC 7. Buffer QF 8. RNase A 2 .1 . 7. b). 1. 2.
BioNick™ Labelling System (Gibco BRL)
lOx dNTP Mix: 0.2mM each dCTP, dGTP, dTTP, O.lmM dATP, O.lmM biotin-14-dATP, 500mM Tris-HCl (pH7.8), 50mM MgCli
lOOmM 6-mercaptoethanol, lOOpg/ml nuclease-free BSA
DNase 1, 50mM Tris-HCl (pH7.5), 5mM magnesium acetate, ImM 6- mercaptoethanol, O.lmM phenylmethylsulphonyl fluoride, 50% (v/v) glycerol, 100|ig/ml nuclease-free BSA
3. Control DNA: 5|ig pBR322 in lOmM Tris-HCl (pHS.O), ImM EDTA
4. Stop Buffer in 300mM EDTA
5. Distilled Water
2 .1 .7 . c). Thermo Sequenase™ fluorescent labelled primer cycle sequencing kit (Amersham)
1. A reagent: Tris-HCl (pH9.5), magnesium chloride, Tween™20,
Nonidet™P-40, 2-mercaptoethanol, dATP, dCTP, dGTP, dTTP, ddATP, thermostable pyrophosphatase and Thermo Sequenase DNA polymerase. 2. C Reagent: Identical to 1. except ddCTP replaces ddATP.
3. G Reagent: Identical to 1. except ddGTP replaces ddATP.
4. T Reagent: Identical to 1. except ddTTP replaces ddATP.
5. Formamide loading dye - Formamide, EDTA, methyl violet.
2 .1.7. d). fm ol DNA Sequencing System (Promega)
1. dATP/ddATP Mix: 350[iM ddATP, 20pM 7-deaza dGTP, 20pM each
dATP, dCTP, dTTP
2. dCTP/ddCTP Mix: 200^M ddCTP, 20pM 7-deaza dGTP, 20|xM each
dATP, dCTP, dTTP
3. dGTP/ddGTP Mix: 30|iM ddGTP, 20^iM 7-deaza dGTP, 20ilM each
dATP, dCTP, dTTP
4. dTTP/ddTTP Mix: 600}iM ddTTP, 20|iM 7-deaza dGTP, 20|iM each
dATP, dCTP, dTTP
5. fm ol ® Sequencing 5x Buffer: 250mM Tris-HCl, pH 9.0, lOmM MgCl2
6. Sequencing Grade Taq DNA Polymerase in: 50% glycerol, lOOmM KCl,
20mM Tris-HCl (pH 8.0), O.lmM EDTA, ImM DDT, 0.5% Tween®, 0.5% Nonidet P40
7. T4 Polynucleotide Kinase lOx Buffer: 500mM Tris-HCl, pH 7.5, lOOmM
MgCl2, 50mM DTT, ImM spermidine
8. T4 Polynucleotide Kinase
9. fmol ® Sequencing Stop Solution: lOmM NaOH, 95% formamide,
2 .1 .7 . e). Magic™ PCR Preps DNA Purification System (Promega)
1. Magic™ PCR Preps DNA Purification Resin - 50ml
2. Direct Purification Buffer
3. Magic™ Minicolumns
2. 1. 8. Oligonucleotides
Amplification of ASNS Exons (chapter 4):
Exon 1 sense 5 -GGT GAT GAT TCC CGA AGA AC-3'
Exon la antisense 5'-GTT CAC CCT TAC TTC GGA GC-3'
Exon 2 sense 5’CAG AGA AAA GCT TAT AGC TT-3'
Exon 3 sense Exon 3 antisense
5'GTG ATT CTC CTA AAT CAC CAA-3’ 5'GTA AAA TTC TGG ATT GCT CTC-3’
Exon 4 sense Exon 4 antisense
5'-GGG AAA ATA GGA ATG TAG ATG-3' 5'-GTG CCA AAT CTT ATG TGT TC-3'
Exon 5 sense Exon 5 antisense
5'GGG AAC ACA TAA GAT TTG GC-3' 5'CCA AAG GGA TGG CTA CAA AT-3'
Exon 6 sense Exon 6 antisense
5'GAC ACT TCC ACT TCC TCT TT-3' 5'GAT TGC AGT AGC ACT AGA TA-3'
Exon 7 antisense 5'GCA CCA CAT CTC TCT AAA CC-3'
Exon 8 sense Exon 8 antisense
5'GAC AAA ATG GTT CCG GGT TA-3' 5'GCA AAC ACT ATC AGA TTC CC-3'
Exon 9 sense 5'GTC TTA TCC ACC TTT AGG AT-3'
Exon 9 antisense 5'G GTA ACA TAT AGC CAA TCA GC-3'
Exon 10 antisense 5'GTT TAC CTA CCT TTG GAA TTC-3’
Exon 11 sense 5 GTG TTA GAA TGG GAT AGA AAA A-3’
Exon 11 antisense 5’A AC GTC TCT CTC AGG AGA TG-3’
Exon 12 antisense 5GAA ACC CAA GTT AGC CTG AG-3'
Exon 1 antisense, exon la sense, exon 2 antisense, exon 7 sense and exon 12 sense could not be obtained.
ETS Primers, for amplification of the ETS gene (used on DNA extracted from SCID- hu chimeras - chapter 7):
ETS + 5 -GAT GAG GTG GCC AGG AGA TG-3’
ETS - 5’-TCA CTC GTC GGC ATC TGG CTT-3'
2 .2 . Methods
2 .2 .1 . Tissue Culture
The cell line DD1027, an EBV-transformed human B-cell lymphoblastoid cell line with karyotype 46,XY, inv(7)(q22.1-34), was obtained from the European Collection of Animal Cell Cultures, Salisbury, UK. This was grown as a suspension in RPMI 1640 with 10% heat inactivated, mycoplasma negative fetal calf serum, 2mM L-glutamine and 100|ig/ml final concentration of gentamicin. Cultures were maintained at a density of l-2x 10^/ml in 75ml tissue culture flasks in a humidified atmosphere containing 5% carbon dioxide at 37®C. The cells were split 1:2 once or twice per week, depending on their growth rate as determined by observation on an inverted light microscope. Manipulation of cells was carried out in a laminar flow hood. For estimation of cell numbers per ml a small aliquot was removed and counted using a haemocytometer counting chamber. Cells were also cryopreserved for long-term storage. Culture medium was poured into a 50ml falcon tube and centrifuged in a Jouan BR311 at 1500rpm for 5 minutes. The pellet was resuspended in 1ml 50% fetal calf
serum, 30% DMSO and 20% RPMI, frozen initially at -70^C and then immersed in liquid nitrogen.
Human patient bone marrow samples were collected into either preservative-free heparin or EDTA-containing tubes and transferred for culture into RPMI 1640 with fetal calf serum, L-glutamine and gentamicin as above. Culture conditions were also as above. Adequate cytogenetic analysis requires a satisfactory number of high quality metaphases for study. Short term culture of human bone marrow samples for up to 72 hours improves the quality and number of divisions seen compared to direct culture and generally does not select for new aberrant clones nor eliminate the original abnormal clone although this can occur especially in myelodysplasia samples (Heim and Mitelman, 1986). Normal human peripheral blood samples were cultured in the same way with phytohaemagglutinin of final concentration 0.05|Xg/ml.
2 .2 .2 . Preparation of Metaphase Spreads
In cases a) and c), synchronization of cultures was carried out to increase the number of cells dividing at the time of arrest of the culture. 1-2 x 10^ cells were resuspended in 10 ml RPMI with additives and after 24 hours, 3mg thymidine (0.5ml of 6mg/ml giving a final concentration of 0.3mg/ml) added. 18 hours later, cells were spun at 1200rpm for 5 minutes, the supernatant discarded and the cells resuspended in lOmls warmed PBS. The wash was repeated and cells were then resuspended in lOmls warmed RPMI containing 50|il bromodeoxyuridine (2mg/ml). After a further 6 hours of culture, lOOp-l colchicine (4|ig/ml giving a final concentration of 0.04pg/ml) was added to halt replication, and left for 10-15 minutes. Colchicine was added in the same way for case b), patient samples which had not been subjected to synchronization. The use of synchronization for patient samples in the study of cancer genetics is controversial due to the possible effects on an abnormal clone and has not been shown to give more informative results in these cases (Testa et al, 1985). Cells were spun at 1200rpm for 5 minutes and resuspended in 10ml hypotonic potassium chloride solution (0.075M). Samples were left to stand at room
temperature, for 10 minutes for cell lines and 12-14 minutes for patient samples. Four drops of fixative solution (3:1 methanol'.glacial acetic acid) were added and the sample spun at 1200rpm for 5 minutes. The pellet was resuspended in 10 ml fixative and spun at 1200rpm for 5 minutes, 3 times. Fixative was added to give a final p ^ e milky suspension.
Microscope slides were washed in hot water with 5 ml decon, shaking, for 30 minutes, then rinsed in cold water for one hour. Slides were kept in 70% ethanol at 4®C. For use, slides were dried and 2 separate drops of cell suspension placed on each slide which were dried in air for at least 24 hours before further use.
2 .2 . 3. Cell Separation
The separation of lymphocytes and polymorphonuclear granulocytes from whole blood can be performed using Polymorphprep™. The solution contains 13.8.% w/y sodium metrizoate and 8% w/v dextran 500. Using a mixture of sodium metrizoate and Ficoll (lymphoprep), polymorphonuclear granulocytes can be centrifuged to the bottom of the tube together with erythrocytes. Polymorphprep™ is a further development of this method. The mononuclear and polymorphonuclear granulocytes are separated into 2 distinct bands free from red cells.
Whole blood obtained from venesection or bone marrow obtained from aspiration was collected into preservative-free heparin or EDTA-containing tubes. 3.5ml of Polymorphprep™ was placed in a 15ml falcon tube and 3.5-5ml anticoagulated blood or bone marrow carefully laid on top. The tube was centrifuged at 1200rpm for 20 minutes at room temperature, in an lEC Centra-7R centrifuge. After centrifugation, 2 leucocyte layers were visible. The top band at the sample/medium interface represented the mononuclear cells and the lower band, the polymorphonuclear granulocytes. The erythrocytes were pelleted. The cell bands could be collected using a Pasteur pipette. The polymorph population was diluted with one volume of 0.45% sodium chloride in order to restore normal osmolality. Cell suspensions could then be washed in RPMI 1640 culture medium,
spun at 1200 rpm for 10 minutes and resuspended in RPMI 1640 and cultured as described in 2.2.1. Alternatively, cells could be subjected to DNA extraction (2.2.5.).
2 .2 . 4. Growth of Cosmids, FACs, YACs and cDNA Clones 2 .2 .4 . a). Cosmids and FACs
Cosmids and FACs were picked from gridded well plates, just thawed from -70®C, or from agar stabs, and plated on to LB agar containing 20|ig/ml kanamycin. After overnight culture at 37®C, a single colony per cosmid or FAC was picked and placed in 20ml liquid LB, containing 20|ig/ml kanamycin. These were cultured overnight at 37^0 in a shaking incubator. The tubes were then spun in a Mistral 3000 centrifuge at 3000rpm for 20 minutes and the supernatant discarded. The pellet was then subjected to a DNA extraction procedure.
2 . 2 . 4.b). YACs
YACs were picked from a YAC plate or stab and plated on to YAC agar and grown at 30°C for a minimum of 48 hours. Single colonies were picked and placed in 20ml YAC broth and cultured overnight at 30®C in a shaking incubator. Tubes were spun in a Mistral 3000 centrifuge at 2000rpm for 5 minutes and the supernatant discarded. The pellet was subjected to DNA extraction.
2 . 2 . 4.C). cDNA
cDNA growth and preparation was identical to that for cosmids except that 75|ig/ml ampicillin was added to the LB agar and liquid broth instead of kanamycin.
2 .2 .5 . Extraction, Purification and Quantification of DNA
It was necessary to extract and purify genomic DNA from blood, tissue suspensions, bacteria-containing cosmids, FACs, YACs and cDNA.
2. 2. 5. a). DNA Extraction From Blood or Bone Marrow
Blood obtained from venepuncture or bone marrow obtained by aspiration was placed in a 50ml universal tube containing 100|il of preservative free heparin. The sample was mixed with 20ml of sterile, cold distilled water to lyse the red cells. The tube was centrifuged at 4000rpm in a Jouan CR412 for 15 minutes and the supernatant discarded. The pellet, containing leucocytes, was resuspended in 30mls of 0.1% Nonidet P40 to lyse the cell membrane but not the nuclear membrane. The tube was spun again at 4000rpm for 15 minutes and the supernatant discarded. The nuclear pellet was resuspended in 10ml of Cell Lysis Buffer (2.1.6.) and incubated overnight at 37®C. The crude DNA solution was purified by phenol/chloroform extraction (2.2.5.f)).
2 .2 .5 . b). DNA Extraction From Tissue Suspension
Samples were treated as in 2.2.5.a). except that the initial lysis of red cells with water was deemed unnecessary.
2 .2 .5 . c). DNA Extraction From Cosmids and cDNA
The pellet obtained from centrifugation (2.2.4.a)) was resuspended in 200|il maxi-buffer (2.1.6.). After 10 minutes on ice, 400}il 1%SDS/0.2M NaOH was added, the mixture left on ice 10 minutes, then 200|il 3/5M potassium acetate added. The mixture was shaken gently and left on ice for 30 minutes. The tube was then spun in a microfuge at 12,000rpm for 10 minutes. The supernatant was transferred to a new tube and kept. 0.6 volumes of isopropanol was added to precipitate DNA. The tube was left at room temperature for 1 hour. The tube was spun at 13,000rpm for 10 minutes and the supernatant discarded. The pellet was washed with 85% ethanol, spun at 13,000 for 2 minutes, the ethanol discarded, the pellet air dried and resuspended in 450|il TE buffer. 10|il RNase A (lOmg/ml) was added and the tube incubated at 37®C for 30 minutes. The sample was then subjected to phenol/chloroform extraction (2.2.5.f)).
2. 2. 5. d). DNA Extraction From YACs
The pellet obtained from centrifugation (2.2.4. b)) was resuspended in 500|ll distilled water and spun at 13,000 for 10 seconds. The supernatant was discarded and to the pellet were added 200|il GDIS (2.1.6.), 200|il phenol:chloroform:isoamyl alcohol (25:24:1) and 0.35g acid washed glass beads (Orme Scientific). The tube was vortexed vigorously for 2.5 minutes, then 200|il distilled water was added. The tube was spun at 13,000rpm for 5 minutes and the aqueous layer transferred to a new tube. 50|Xg RNase A (lOmg/ml) was added, the tube incubated at 37®C for 30 minutes and then subjected to phenol/chloroform extraction (2.2.5.f))-
2. 2 .5 . e). DNA Extraction From PACs
The method initially followed that for cosmids until the resuspension in TE buffer. The pellet was resuspended in 3ml TE buffer and 1ml lOM ammonium acetate and left on ice for 20 minutes. The tube was spun in a microfuge at 13,000 for 5 minutes and the supernatant kept. DNA was precipitated by adding 2.5 volumes of 100% ethanol, spinning at 13,000 for 5 minutes and resuspending the pellet in 500|xl TE buffer. 10|il RNase A (lOmg/ml) was added and the tube incubated at 37°C for 30 minutes. The sample was then purified through a Qiagen column (2.1.7.a)). The volume was made up to 1ml by making the final concentration 750mM NaCl and 50mM MOPS. The column was prepared in a