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Found 3 matching solutions for this experiment
Upstream tips |
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Agarose plugs were cut with a sterile scalpel to fit the size of the combs of the gel casting (3-4 mm). They were washed in restriction buffer at 4°C for 30 minutes. The restriction enzymes (REs) used for PFGE sample preparation included DraI and XbaI (Fermentas, Canada) with the appropriate restriction buffer. The restriction digestion steps were done separately by adding 30 U of XbaI enzyme followed by incubation at 37°C for four hours and DraI enzyme followed by an overnight incubation at 37°C. |
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Protocol tips |
Agarose plugs were cut with a sterile scalpel to fit the size of the combs of the gel casting (3-4 mm). They were washed in restriction buffer at 4°C for 30 minutes. The restriction enzymes (REs) used for PFGE sample preparation included DraI and XbaI (Fermentas, Canada) with the appropriate restriction buffer. The restriction digestion steps were done separately by adding 30 U of XbaI enzyme followed by incubation at 37°C for four hours and DraI enzyme followed by an overnight incubation at 37°C. |
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A region in intron 6 of CYP2E1 (7632T>A) was amplified and digested with DraI (8U, New England BioLabs). Wild type CYP2E1 allele (*1A) has restriction sites for RsaI and DraI, but not for PstI. The presence of restriction sites yielded two fragments of 352 and 143 bp for RsaI restriction digest, 377 and 118 for PstI, and 268 and 306 for DraI restriction digest. |
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Protocol tips |
A region in intron 6 of CYP2E1 (7632T>A) was amplified and digested with DraI (8U, New England BioLabs). Wild type CYP2E1 allele (*1A) has restriction sites for RsaI and DraI, but not for PstI. The presence of restriction sites yielded two fragments of 352 and 143 bp for RsaI restriction digest, 377 and 118 for PstI, and 268 and 306 for DraI restriction digest. |
Upstream tips |
Protocol tips |
Downstream tips |
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The restriction endonucleases BamHI, BstPI, DraI, EcoRI, Sau3AI, and SmaI (Takara Shuzo Co.); DpnI, HincII, and HindIII (New England BioLabs); and EcoRV (Nippon Gene) were used. The reaction conditions for these enzymes were as recommended by the suppliers |
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Protocol tips |
The restriction endonucleases BamHI, BstPI, DraI, EcoRI, Sau3AI, and SmaI (Takara Shuzo Co.); DpnI, HincII, and HindIII (New England BioLabs); and EcoRV (Nippon Gene) were used. The reaction conditions for these enzymes were as recommended by the suppliers |
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