Bio-Rad SsoAdvanced™ Universal SYBR® Green Supermix - Manual (Page 8)

2 | SsoAdvanced ™ Universal SYBR ® Green Supermix Instruction Manual 2 |
DNA Samples
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Isolate DNA using the appropriate method for the given sample type (for example, column
purification for cell lines, phenol/chloroform or column purification for tissue samples)
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Store the DNA in an appropriate solution
– 0.1 mM EDTA (in DEPC-treated ultrapure water)
– TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.0)
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Store the DNA at –80ºC in single-use aliquots
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Assess DNA quality with an agarose gel; a single band indicates high integrity DNA, whereas
a smear indicates degraded DNA
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Assess the DNA purity using a spectrophotometer for the following:
– A260/A230 >1.5 (lower ratios may be attributed to carryover guanidine, and/or inhibitors
like humic acid and organics)
– A260/A280 1.7–2.0 (lower ratios are indicative of contaminants from salts, carbohydrates,
peptides, proteins, phenols, and guanidine thiocyanate)
– Higher ratios may be indicative of RNA contamination
Tips:
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Heat treating DNA may be required prior to qPCR to relax strong secondary structure
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Using a restriction digest enzyme may be required for select qPCR applications, such as copy
number variation, to reduce signal-to-noise ratio.
Plasmid Samples
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Prepare plasmids using an appropriate method
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Store the stock plasmid in an appropriate solution
– TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
■ ■
Store the plasmid at –80ºC in single-use aliquots
■ ■
Assess plasmid quality with an agarose gel; a single band indicates high integrity plasmid,
whereas a smear indicates degraded plasmid or excess enzymatic activity
■ ■
Assess the plasmid purity using a spectrophotometer for the following:
– A260/A280 1.7–1.9 (lower ratios are indicative of contaminants from salts, carbohydrates,
peptides, proteins, phenols, and guanidine thiocyanate)
– Higher ratios may be indicative of RNA contamination