1.10 ANÁLISIS DE REQUERIMIENTOS [13]
1.10.6 SISTEMA DE CONTROL DE HORARIOS (SICOH)
Solutions were made up in Milli-RO plus (dH20)or Milli-Q plus (ddHzO) water and then autoclaved at 15 Ib/sq. in. at 121 °C for 20 minutes.
Alkaline lysis buffers
Solution I
50mM glucose; 25mM Tris-Cl (pH 8.0); lOmM EDTA (pH 8.0) Autoclave at 10 Ib/sq. in. for 15 minutes and store at 4°C. Solution II
0.2M NaOH, freshly diluted from lOM stock; 1% SDS Solution III
60ml 5M KAc; 11.5ml glacial acetic acid, made up to lOOmls with dH20. (3M with respect to potassium and 5M with respect to acetate)
Bonding solution
3ml 95% ethanol; 5pi y-methocyloxylpropyltrimethoxy silane; 50pl 10% acetic acid.
Church prehybridization/hybridization buffer
0.5M NaPi (pH 7.2); 7% SDS; ImM EDTA
Denaturing solution
0.5MNaOH; 1.5MNaCl
Herring/salmon sperm DNA
DNA was dissolved to a concentration of lOmg/ml and then autoclaved for 5 minutes to shear the DNA. DNA fragments should be 500-Ikb in length.
6 X Loading buffer (agarose gel)
0.25% xylene cyanol; 0.25% bromophenol blue; 30% glycerol
Loading buffer (formamide)
98% v/v formamide; 0.3% bromophenol blue; 0.3% xylene cyanol; lOmM EDTA
Neutralizing solution
1.5M NaCl; 0.5M Tris-Cl (pH 7.5)
20 X SSC
150mM NaCl; 15mM Na Citrate (pH 7.0)
10 X TEE
0.89M Orthoboric acid; 0.89M Tris ; 20mM EDTA
TE
TNE/SDS
IM Tris-Cl (pH 7.5); 5M NaCl; 0.5M EDTA; 0.1% SDS.
2.1.1.1 Solutions for RNA work
All solutions used for RNA work were prepared with DEPC-treated water unless stated otherwise. If possible, previously unopened chemicals were used for the preparation of solutions. Cuvettes used for spectrophotometry were filled with 1:1 HCl:Methanol for 1 hour and then rinsed thoroughly in DEPC-H2O before use.
DEPC-treated water
Diethyl pyrocarbonate was added to Milli-RO plus water at a concentration o f 0.1%. The water was left to stand at 37°C for 2 hours and then autoclaved.
10 X MOPS/EDTA buffer (Northern electrophoresis buffer)
0.2M MOPS; 50mM NaAc; lOmM EDTA (pH 7.0).
RNA loading buffer
0.75ml deionised formamide; 0.3ml 5 x Northern electrophoresis buffer; 0.24ml formaldehyde; 0.1ml glycerol; 0.08ml 10% bromophenol blue.
3M LiCl/6M urea
63.6g LiCl; 180.2g urea made up to 500mls with DEPC-treated water. Not autoclaved; store at 4°C.
lOmM Tris pH 7.5/0.5% SDS
1ml IM Tris pH 7.5; 5 mis 10% SDS made up to lOOmIs with ddHzO.
20 X PBS
23g Na2HP04, 4g KCl, 160g NaCl, 4g KH2PO4, l l g Na Butyrate made up to 800mls with distilled water. Autoclave and add 200mls 0.5M EDTA.
2.1.1.2 Bacterial cell culture Ampicillin
lOOmg/ml in ddH20, filter-sterilized through a 0.2pm filter. Used at a working concentration o f lOOpg/ml. Stored at -20°C.
IPTG
Stock solution made up at lOOmM, filter-sterilized through a 0.2pm filter. 4pl o f the stock was spread on a 90mm agar plate (with the appropriate antibiotic) and left to absorb for 30 minutes. Stored at -20°C.
Kanamycin
25mg/ml in ddH20, filter-sterilized through a 0.22pm filter. Used at a working concentration o f 25pg/ml. Stored at -20°C.
L broth/agar and LM broth
1.6g tryptone, 1 Og yeast extract, 0.5g NaCl, made up to lOOmIs with dH20. Autoclaved. For LM broth, 0.2g o f MgSO4.7H20 was added. For L agar, 2.0g o f bacto-agar was included.
L top agarose
0.5g tryptone, 0.25g yeast extract, 0.5g NaCl, 0.35g agarose, made up to 5Omis with dH20. Autoclaved.
20% Maltose
lOg of maltose made up to 50mls with ddHzO. Filter-sterilized through a 0.22pm filter. Stored at 4°C.
SM buffer
5.8g NaCl, 2g MgSO4.7H20, 6.05g Tris base, 5mis 2% gelatin made up to 11 with dH20. Adjust to pH 7.5 with HCl. Autoclaved.
Tetracycline
5mg/ml in ethanol. Used at a working concentration o f 5pg/ml. Stored at -20°C.
2 X TY broth/agar
Ig tryptone, Ig yeast extract, 0.5g NaCl made up to lOOmIs with dH20. Autoclaved. For 2 X TY agar, 1.5g of bacto-agar was included.
X-Gal
Stock made up to 20mg/ml in dimethylformamide. 40pl o f the stock was spread on a 90mm agar plate (with the appropriate antibiotic) and left to absorb for 30 minutes. Stored at -20°C.
2.1.1.2.a Bacterial strains Y1088
//5£/R(rk’mk ), TMcrB , metB, proC :: Tn5, supE, supE, to n A ll, trpK, A/acU169, F-, X-
(pMC9) note: pMC9 = pBR322-/ac 1**, Tn5 confers kan\ pMC9 confers amp^and tef.
Used for immunological screening o f expression libraries and propagation o f X gtll and Xgt 18-23. Expression o f the foreign protein is controlled by the high levels o f lac
repressor made by the resident plasmid pMC9, which carries lac 1**. The supV marker allows for cell lysis which facilitates antibody screening.
2.1.2 cDNA library
The human adult testis cDNA library was purchased from Clontech Laboratories (catalogue no. HLlOlO). The library was constructed in the vector Lambda g t l l in the
EcoR l cloning site; the original estimated titre o f the library was 9.8 x 10^ pfu/ml; before use the library was re-titred and found to be 7.5 x 10* pfu/ml. The average insert size was 1.2kb.
2.1.3 Suppliers of materials
2.1.3.1 Chemicals, reagents and membranes
All chemicals were o f AnalaR quality and purchased from British Drug House (BDH) unless stated otherwise.
0.24-9.5kb RNA ladder GibcoBRL
redivue a^^P-dCTP (3000Ci/mmol) 40% 19:1 acrylamide:bisacrylamide agarose chloroform dNTPs HybondN/lsr membranes Lambda DNA Lymphoprep™
Kodak biomax MR X-ray film Mineral oil Northern blot p AMP 10 plasmid pd(N)6 random hexamers phenol proteinase K RNAzol™ B Amersham Severn Biotech. Sigma Amresco Pharmacia Amersham
New England Biolabs Nycomed, Oslo, Norway Anachem Sigma Clontech Gibco BRL Pharmacia Amresco Amresco Biogenesis 2.1.3.2 Kits Lambda maxi kit
LigATor PCR cloning kit Microprep mRNA kit Rediprime labelling kit Sequenase Version 2 kit TA PCR cloning kit
Wizard™ miniprep DNA purification kit Wizard™ PCR prep DNA purification kit
Quiagen R&D Systems Pharmacia Amersham Amersham Invitrogen Promega Promega 2.1.3.3 Enzymes DNAse Klenow
MMLY reverse transcriptase Proteinase K
Restriction enzymes RNAse inhibitor Tag polymerase (5u/|al)
Red Hot Tag polymerase (5u/pl)
Pharmacia Boehringer
Pharmacia or Advanced Biotechnologies Boehringer
New England Biolabs Pharmacia
Promega
Advanced Biotech
2.1.3.4 Acknowledgements for materials
The human brain RNA was fi'om Dr. A. Bennett, University of Nottingham. The human liver, lung, placenta and muscle tissues were from Prof. S. Povey, MRC HBGU, UCL. The somatic cell hybrids used were from the Galton Laboratory Somatic Cell Hybrid Panel, a gift from S. Jeremiah and Prof. S. Povey.jHealthy tissue was used from a testicular
cancer patient (a gift from Dr J. Round). 2.1.4 Oligonucleotides
All oligonucleotides were synthesised by Oswel DNA service, Genosys or supplied with kits.
For amplification o f inserts cloned in pCRII from the TA PCR cloning kit:
T7 ATACGACTCACTATAGGG
SP6 TTTAGGTGACACTATAG
M13 (-20) TAAAACGACGGCCAGT or
M l 3 universal TAAAACGACGGCCAGT
M13 reverse ACAGCTATGACCATG
For amplification o f inserts cloned in pTAg from the ligATor PCR cloning kit:
pTAg 5' CTATGACCATGATTACGCCAA
pTAg 3' GTAAAACGACGGCCAGTGAA
M l 3 primers can also be used.
For amplification o f inserts cloned in Xgtl 1 :
Xgtl 1 Forward GGTGGCGACGACTCCTGGAGCCCG
A,gtl 1 Reverse TTGACACCAGACCAACTGGTAATG
For amplification o f DDRT cDNAs:
4VA2 Forward AAAACTGCAGCACTTCTGAC
4VA2 Reverse CTGTCAGCCTCTCCACATTG
lOVCl Forward ATCATTGCATGATAGAAGAGC
lOVCl Reverse AAATGCTTGGCAATGTTAAATG
13VA2 Forward TTTTTCACACATAACCACTGCC
13 VA2 Reverse GAGAGGGAAGAGTGAAGCTCC
For amplification o f MLcDNAs:
M LIGI Forward CAGGGTGGAGAACCTTCAAA
M LIGI Reverse GGCTGTCATTTCTCCACCTA
For amplification o f SRY:
SRY Forward GAAT ATTCCCGCTCTCCGG
^ T R e v e rse GCTGGTGCTCCATTCTTGA
For amplification o f PGM.
PGM Forward AAC AAGATGCCCTTGGGAGCTGTGA (in exon 2)
PGM Reverse GAACTGATTGGAC AGAAGGC ACT AG (in exon 4)
DDRT-PCR primers: See section Al.1.2