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Gel casting tray Sample comb Electrophoresis chamber & power supply Agarose  (creating pores)  Electrophoresis buffer  [1X  TAE  ( Tris  acetate EDTA) or 1X TBE( Tris  borate EDTA)] Loading buffer  (containing  glycerol  &  bromophenol blue ) Ethidium bromide  (intercalates between bases of nucleic acids) UV  transilluminator (to visualize nucleic acid stained with ethidium bromide)

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Procedures for 1 % agarose gel preparation

Agarose (1 g) + Buffer (100 ml)  Leave to cool + Eth Br Pour in tray Remove comb after polymerization Loading samples and electrophoresis Visualize under UV transilluminator

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http://bristoluniversityfacultyofscience.blogspot.com/2010_06_01_archive.html http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html

DNA separation by  Agarose  gel electrophoresis

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As DNA molecules are negatively charged (because of phosphate groups), negative pole is connected near samples

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100bpLadderDNAM±r
(1 m bp-2000bp)
bp
1500
100
ng/loul
Real Biotech Corportion

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G:\kolya\fourth year genetics\DNA isolation 23-9-2012.tif

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Effect of increasing Agarose concentration

Higher concentrations of agarose facilities separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

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DNA concentration  α  amount of absorbance 1 absorbent (O.D.) at 260 nm = 50  ng /µl (µg/ml) of  d.s . DNA 					       = 33  ng /µl  s.s . DNA 					      = 40  ng /µl RNA Dilution factor = total volume/added volume of DNA  DNA concentration of sample = A260 x 50 x dilution factor

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A260: max absorbance for nucleic acid A280: max absorbance for proteins DNA purity= A260/A280= 1.7-2 If less, therefore protein contamination If more, therefore RNA contamination

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10 µl were taken from a sample to measure its concentration and purity; 	A260 nm= 0.122 		&     A280= 0.093 	a 3 ml  cuvette  was used and distilled water was used as a blank.  Calculate DNA concentration in the provided sample per µl.  Answer?? Sample  concn = 0.122x50x(3000/10)= 1830  ng /µl Purity= A260/A280= 0.122/0.093= 1.3

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Diphenylalanine , binds with DNA, (blue dye) at  = 600 nm Standard curve is used.

0.8
0.6
0.4
02
1500
2000
Conæntration [ng/pL]

Absorbance of sample of unknown concentration

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How to make a standard curve to measure DNA concentration

Data for known DNA concentrations are used to make the standard curve, plotting concentration on the X axis, and the absorbance on the Y axis. The same assay is then performed with samples of unknown concentration.  To analyze the data, one locates the measurement on the Y-axis that corresponds to the assay measurement of the unknown substance and follows a line to intersect the standard curve. The corresponding value on the X-axis is the concentration of substance in the unknown sample.

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https://www.youtube.com/watch?v=vtxb6Tr8Y3s https://www.youtube.com/watch?v=EZjNuqSEPbY https://www.youtube.com/watch?v=vq759wKCCUQ https://www.youtube.com/watch?v=dedL1Br3Nos

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Gel Electrophoresis 

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 https://www.youtube.com/watch?v=vtxb6Tr8Y3s https://www.youtube.com/watch?v=EZjNuqSEPbY https://www.youtube.com/watch?v=vq759wKCCUQ https://www.youtube.com/watch?v=dedL1Br3Nos

Gel Electrophoresis

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