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1
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Lysis Buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA, Tween 20:0.5%)
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Used to resuspend placenta samples for DNA isolation.
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30821558
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2
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Proteinase K (20 mg/ml stock solution)
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Treated placenta samples overnight at 37°C for protein digestion during DNA isolation.
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3
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Phenol-chloroform
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Used for DNA extraction from digested placenta samples.
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4
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Ethanol
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Used to precipitate DNA in the DNA isolation process.
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5
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TE Buffer (Tris 1.1 g, EDTA 0.37 g, dd H20 1000 ml, pH 8.1)
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Used to dissolve the extracted DNA after phenol-chloroform extraction.
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6
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PCR Buffer (10x, 5 ml)
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Used in the PCR reaction mixture for amplifying DNA.
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7
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MgCl2 (25mM, 1 ml)
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Included in the PCR reaction mixture to facilitate DNA polymerase activity.
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8
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dNTP mix (100 mM, 1 ml)
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Provides the necessary nucleotides for DNA synthesis during PCR.
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9
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Primers (PCO3: 5’-ACACA ACTG TGTTC ACTAGC-3’ and PC04:5’-CAA CTT CATCCAC GTTCACC-3’)
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Used to amplify the β-globin gene for confirming DNA quality.
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10
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HotStart Plus DNA Polymerase (Qiagen, 0.5 ml)
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Used in the PCR reaction mixture to synthesize DNA.
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11
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Primers (AAV-F1: 5’-ACC AAC ATC GCG GAR GCC AT-3’; Ra: 5’-GGC TGC TGG TGT TCR AAG GT-3’; Rb: 5’-GGC TGC TGA TGC TCG AAG GT-3’)
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Used to amplify AAV DNA in the primary round of hemi-nested PCR, yielding a 297 bp fragment.
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12
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Primers (F2: 5’-AAC TGG ACC AAT GAR AAC TTT CCC T-3’; Ra: 5’-GGC TGC TGG TGT TCR AAG GT-3’; Rb: 5’-GGC TGC TGA TGC TCG AAG GT-3’)
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Used in the second round of hemi-nested PCR to amplify a 247 bp AAV DNA fragment.
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13
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Agarose Gel (2%)
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Used in electrophoresis to resolve amplified PCR products.
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14
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Safe Stain
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Stains DNA in agarose gel for visualization under UV light.
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