| Basic Protocol 1: Restriction Fragment Length Polymorphism (RFLP)–Southern Blot for Genomic DNA Analysis of C4 Gene Dosage Materials -
High‐molecular‐weight human genomic DNA, prepared from peripheral blood using commercial kit (e.g., Puregene; Gentra Systems) -
10 U/µl restriction endonucleases (Taq I or Psh AI and, for Psh AI‐Pvu II double digest only, Pvu II) and appropriate buffers (New England Biolabs) -
Mineral oil (optional) -
High‐gelling‐temperature agarose, biotechnology grade (Amaresco) -
1× TBE (see recipe ) -
0.5% (v/v) ethidium bromide -
Glycerol dye (see recipe ) -
Depurination buffer: 0.2 M HCl/0.5 M NaCl, optional -
Denaturation buffer: 1.5 M NaCl/0.5 M NaOH -
Neutralization buffer: 1.5 M NaCl/0.5 M Tris·Cl, pH 7.4 -
10× SSC: 1.5 M NaCl/0.15 M sodium citrate -
SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe ), 42°C -
20 mg/ml sheared salmon or fish sperm DNA (ssDNA; Amaresco) -
Prehybridization solution (see recipe ) -
Appropriate radiolabeled hybridization probes (see protocol 3 ) -
0.1% (w/v) SDS/2× SSC -
0.5% (w/v) SDS/0.1× SSC -
Thermal cycler with heated top or water bath set at 65°C, for Taq I digest only -
25° and 37°C water baths for Psh AI and Psh AI‐Pvu II double digest, respectively -
Horizontal agarose gel apparatus (e.g., model GNA200; Pharmacia) and constant power supply (e.g., model 3000; Pharmacia) -
1‐liter conical flask -
Photodocumentation machine with UV transilluminator -
Nylon membrane (e.g., Hybond N+; Amersham Biosciences) cut to exact size of gel (e.g., 20 cm × 20 cm) -
Two pieces Whatman 3MM filter paper cut to exact size of gel (e.g., 20 cm × 20 cm) and additional pieces for supporting membrane -
Blotting device (e.g., Possiblot machine; Stratagene) -
UV cross‐linker (e.g., Stratalinker 2400; Stratagene) -
Hybridization oven including hybridization tubes (e.g., Isotemp; Fisher Scientific), 42°C -
Boiling water bath -
Water bath, with shaking (e.g., Techne), 65°C -
Plastic wrapper (e.g., Clingfilm; Fisher Scientific) -
X‐ray film and film developer (X‐OMAT; Kodak) -
Phosphorimager (e.g., Molecular Dynamics, Amersham Biosciences) and accompanying software (e.g., STORM; Amersham Biosciences), optional Basic Protocol 2: Long‐Range Mapping Pulsed‐Field Gel Electrophoresis (PFGE) for Determination of the Modular Structure of RP‐C4‐CYP21‐TNX (RCCX) Materials -
Very‐large‐molecular‐weight genomic DNA in low‐gelling‐temperature (LGT)‐agarose plugs (see protocol 4 ) -
TE buffer, pH 7.4 ( appendix 2A ) -
5 U/µl Pme I or Pac I restriction enzyme and appropriate buffer -
Agarose -
0.5× TBE (see recipe ) -
λDNA ladder and MidRange Marker II (New England Biolabs) molecular weight markers -
0.5% (w/v) InCert LGT agarose (FMC), molten -
0.5% (v/v) ethidium bromide -
Depurination buffer: 0.2 M HCl/0.5 M NaCl, optional -
Denaturation buffer: 1.5 M NaCl/0.5 M NaOH -
Neutralization buffer: 1.5 M NaCl/0.5 M Tris·Cl, pH 7.4 -
10× SSC: 1.5 M NaCl/0.15 M sodium citrate -
SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe ), 42°C -
20 mg/ml sheared salmon or fish sperm DNA (ssDNA; Amaresco) -
Prehybridization solution (see recipe ) -
Radiolabeled hybridization probe E (Table 13.8.1 ; see protocol 3 ) -
0.1% (w/v) SDS/2× SSC -
0.5% (w/v) SDS/0.1× SSC -
Scalpel, sterile -
2‐ml microcentrifuge tubes -
Pulsed‐field gel electrophoresis system (e.g., CHEF Mapper XA; Bio‐Rad) -
Glass hooks made from Pasteur pipets, sterile -
Photodocumentation machine with UV transilluminator -
Nylon membrane (e.g., Hybond N+; Amersham Biosciences) cut to exact size of gel -
Two pieces Whatman 3MM filter paper cut to exact size of gel and additional pieces for supporting membrane -
Blotting device (e.g., Possiblot machine; Stratagene) -
UV cross‐linker (e.g., Stratalinker 2400; Stratagene) -
Hybridization oven including hybridization tubes (e.g., Isotemp; Fisher Scientific), 42°C -
Boiling water bath -
Water bath, with shaking (e.g., Techne), 65°C -
Plastic wrapper (e.g., Clingfilm; Fisher Scientific) -
X‐ray film and film developer (X‐OMAT; Kodak) Support Protocol 1: Probe Labeling and Desalting for Southern Blots Materials -
Probe DNA (Table 13.8.1 ): restriction fragments or PCR‐amplified DNA fragments, gel purified (unit 10.5 ) -
Multiprime DNA labeling kit (e.g., GibcoBRL Random Primer DNA Labeling System; Invitrogen Life Technologies) containing dNTPs, premix buffer, and Klenow enzyme -
[α‐32 P]dCTP (3000 Ci/mmol; Amersham Biosciences) -
Microspin G‐50 column (Amersham Biosciences) -
SDS‐SET solution: 0.1% (w/v) SDS in SET solution (see recipe ) Support Protocol 2: Preparation of Genomic DNA Plugs from Unicellular Nucleated Cells Materials -
Ficoll‐Paque Plus solution (Amersham Biosciences) -
EDTA or heparinized whole blood ( appendix 3F ) -
PBS (see recipe ), 4°C -
1% InCert low‐gelling‐temperature (LGT) agarose (FMC): boiled, divided into aliquots in 2‐ml microcentrifuge tubes, and equilibrated at 60°C -
NDS solution (see recipe ) -
1.5 mg/ml proteinase K (Fisher) -
15‐ml centrifuge tubes -
Refrigerated tabletop centrifuge (e.g., Eppendorf 5810R), 4°C -
Plug molds (Bio‐Rad), bottom openings sealed with Scotch tape -
2‐ml microcentrifuge tubes -
Glass hooks made from Pasteur pipets, sterile -
50°C water bath Alternate Protocol 1: Module‐Specific PCR for Determination of the Number of RP‐C4‐CYP21‐TNX (RCCX) Modules or Total C4 Gene Dosage Materials -
Control genomic DNA samples from individuals with three, four, and five C4 genes -
100 ng/µl oligonucleotide primers: -
b: 5′‐GCT CAA GCT GTG AGG AGA ACT‐3′ -
c: 5′‐TAT CAC AGG CTC TGG CCC CA‐3′ -
d: 5′‐TTC GTG GTC CAG TAC AGG GA‐3′ -
Deionized H 2 O, autoclaved -
Platinum Taq PCR x DNA Polymerase (Invitrogen; or equivalent commercial high‐fidelity Taq polymerase kit), including: -
2.5 mM dNTP mix: 2.5 mM each dNTP in TE buffer, pH 7.5 ( appendix 2A ), store at −20°C -
Genomic DNA of interest -
Positive control DNA with bimodular (B)/monomodular (M), B/B, and trimodular (T)/B RCCX modules (Fig. ) -
Thermal cycler and appropriate PCR tubes -
Photodocumentation machine with UV transilluminator -
Image quantification software (e.g., ImageQuant TL; Amersham Biosciences) -
Linear regression software (e.g., Excel; Microsoft) NOTE: The steps are written for primers b, c, and d ( TNXA‐RP2/TNXB ). The same method can be used with primers a, b, and c ( RP1 / TNXA‐RP2 ). NOTE: The success of this experiment critically relies on the concentrations of the primers because the intensity of each band will need to represent faithfully the actual dosage of the gene segments. For each new batch of primers, this titration needs to be repeated to ensure that the experiment is working. Alternate Protocol 2: Labeled‐Primer Single‐Cycle DNA Polymerization (LSP) Reaction Coupled with Restriction Fragment Length Polymorphism (RFLP) for Determination of C4A/C4B Gene Dosages Materials -
Commercial PCR amplification kit (e.g., FailSafe PCR System; Epicentre Technologies) -
Genomic DNA of interest -
Forward primers (choose one): -
-
Commercial PCR purification kit (e.g., Amicon Microncon‐PCR Centrifugal Filter Devices; Fisher) -
[γ‐32 P]E29.3 primer (see protocol 7 ) -
Deionized water, autoclaved -
10 U/µl Psh AI or 5 U/µl Xcm I restriction enzyme and appropriate 10× buffer -
Glycerol dye (see recipe ) -
High‐gelling‐temperature agarose, biotechnology grade (Amaresco) -
Thermal cycler -
Scalpel -
Plastic wrapper
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