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By: Ashneet Biln and Carling Gales
DNA Extractions By: Ashneet Biln and Carling Gales
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Introduction DNA is extracted for many reasons, such as medicine, crime investigations, missing persons, understanding genetics and genetic modification, among others. The number of repeats at a certain location can be determined from an extracted sample of DNA, allowing the comparison between two sets of DNA.
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Objectives We did three different DNA extractions this semester, using tissue samples from pork. They were Chelex, salt, and Invitrogen. Once all the DNA samples had been obtained we quantified them using a spectrophotometer. Then using gel electrophoresis we determined the quantity in each of the samples we extracted.
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Chelex Using 5% Chelex solution Involves an enzyme called Proteinase K
Incubate at 55 degrees Celsius Boiling water bath -resin that binds cellular material together because it has an affinity for heavy metal cations (Ca2, Mg2, Mn2) and isolates DNA -enzyme that denatures, and breaks down protein -lysis -open up cells
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Salt 1x lysis (TNES) buffer Enzyme Proteinase K
Incubate at 55 degrees Celsius 5M NaCl 80% Isopropynol 70% ethanol 1x TE buffer Removes Mg ions, detergent removes lipids in cell membrane, breaking it open denatures protein NaCl neutralizes negative DNA, so it’s not extracted with Mg ions Ethanol precipates DNA and removes salts and proteins TE buffer allows resuspension of DNA, preserves DNA b/c it contains EDTA metal chelating agent
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Invitrogen PureLink Genomic Digestion Buffer
Incubate at 55 degrees Celsius RNase A PureLink Genomic Lysis/ Binding Buffer 95% ethanol Wash Buffer 1 Wash Buffer 2 PureLink Genomic Elution Buffer -breaks down protein -Rnase breaks down RNA -lysis/binding buffer binds DNA sample to the filter -ethanol removes salt and proteins -wash buffers remove proteins and salts -elution buffer remove DNA from the membrane so it can be filtered through into the collection tube when centrifuged
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Quantification Spectrophotometer Cuvette DNA is read at 260nm
RNA is read at 280nm Cuvette accuracy read at 320nm Concentration = (OD260 – OD320) x dilution factor x OD constant Reliable Reads the absorption of light at certain wavelengths Put sample into cuvette to be read by the machine Concentration = (OD260 – OD320) x dilution factor x OD constant (dilution factor 10, OD constant 50) Less than 1.8 protein cont., greater than 2.0 organic cont. If OD260 too low, prepare sample with a smaller dilution for a more reliable reading
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Results from Spectrophotometer
Carling Extraction 260nm 280nm 320nm Concentration Chelex 0.8619A 0.662A 0.673A -27ug/mL Salt 0.073A 0.056A 0.051A 11ug/mL Invitrogen 0.022A 0.024A 0.037A -7.5ug/mL Ashneet Extraction 260nm 280nm 320nm Concentration Chelex 2.208A 1.897A 0.165A 1021.5ug/mL Salt 0.073A 0.056A 0.051A 11ug/mL Invitrogen 0.026A 0.027A -0.5ug/mL -
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Results from Spectrophotmeter
Ratio = OD260/OD280 Protein contamination Name Chelex Salt Invitrogen Carling 0.935 1.304 0.595 Ashneet 1.164 1.30 1.00 -tells if sample is viable (protein or organic contamination again) -protein contamination because all less than 1.8
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Agarose Gels 1.5% agarose gel Orange G loading buffer
Gel Star Flourescent Dye TBE running buffer DNA ladder Time and voltage DNA migration ADD COLOUR PIC -2.25g agarose into 150mL of loading buffer b/c big boat -microwaved at 30 second intervals until clear -waited til cool to touch -instructor adds red gel flourescent dye Add combs to boat add gel and allow to solidify/cool (will be opaque) add running buffer WHILE cooling we added 5uL of orange G loading buffer to DNA samples loaded 13 uL of samples into the gel DNA ladder in first well of each row 96V, for an hour DNA negative so migrate to positive end Smaller NDA migrate faster because agarose gel is porous, so smaller pieces fit through faster
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Results of Gel Electrophoresis
Bands Smears Well 6 Invitrogen Experimental Well 9 Chelex Experimental Well 10 Invitrogen Experimental Row 1 CARLING : Well 1 – ladder, well 2 – Chelex control, well 3 – Chelex exp., well 4 – salt control, well 5 – salt exp., well 6 – invitrogen exp., well 7 – invitrogen control ASHNEET: well 8 – chelex control, well 9 – chelex exp., well 10 – invitrogen exp. Looking for a single band that can be compared to the ladder Smear mean DNA is degraded, has been chopped up into all different sixes so migrates quickly and leaves a “smear” WELL 6: looks like the 6th band down so 400bp and 4ng/mL WELL 9: looks like 6th band down so 400bp and 4ng/mL WELL 10: looks like the 4th band down so 600bp and 6ng/mL
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Conclusion The most time efficient method of DNA extraction was the Chelex extraction. However, the most accurate method of DNA extraction was the Invitrogen Extraction. This is apparent from our results, as it was the extraction that showed most clearly when we performed the gel electrophoresis.
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