3. Shown below is an image of a common commercially available IkB DNA ladder (pr

Question

3. Shown below is an image of a common commercially available IkB DNA ladder (produced by Thermofisher). The concentration of DNA within this ladder is 0.Sugul. The amount of each band found within 0.5ug of the ladder is shown in the middle column (ng/0.5ug) along with the percent of the total shown in the last column. Given this information answer the following questions GeneRuler 1 kb DNA Ladder O'GeneRuler 1 kb DNA Ladder bpng 0.5 pg 10000 300 60 2000 250 1500 250 50 1000 60.0 12.0 750 250 50 500 25.0 50 250 250 50 0.5 potane, 8 cm length gel, X TAE, 7 Vcm, 45 min Inspection of the 250bp band and the 2500bp band reveals that the same amount of DNA (25ng) leads to a "brighter" band at 2500bp relative to the band at 250bp. Assuming this gel was stained with an "Intercalating dye (like SYBR safe or ethidium bromide) how can you explain this data? (2.5 points) Why is it important, when quantitating DNA using a gel to compare sample bands to similar sized bands in the standard? (25 peints) a. b. c. In an agarose gel experiment you have loaded lul of the ladder shown above. In the same gel you loaded 5 uL of a linearized plasmid consisting of 5000bp of DNA. Analysis demonstrates that the linearized plasmid band is 2x's as "bright" as the standard band in the gel. What is the concentration of the DNA in linearized plasmid sample? (5 points)

Explanation / Answer

Electrophoresis is a technique used to separate DNA sample on the basis of Charge /mass ratio. It can be used to estimate the different fragments in DNA sample and their respective size.

1) Same amount of DNA gives a brighter band at 2500bp as compared to band at 250bp. This is because although the concentration of DNA is same, The DNA at 2500bp is much longer. It allows more Ethidium bromide and SYBR to intercalate between the bases of DNA. This allows more illumination and more brightness of band.

EtBr and SYBR are intercalating dyes which enter the major grooves of DNA. They help in visualization of DNA in electrophoresis gel.

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2) It’s important to compare DNA fragments of same sizes to the standard ladder. The prime reason is to predict the size of the DNA fragment. DNA fragments of same size will travel similar distances on the gel. By comparing the band to the corresponding band in standard ladder we can get an estimate of the size of DNA fragment.

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3) Given- The size of linear DNA =5000bp

Quantity loaded = 5microlitre

From the Standard ladder with concentration 0.5microgram/microlitre

            Amount of DNA in 5000bp band of ladder =30 ng (from image)

NOW, since the DNA sample has twice the intencity,therefore

Concentration of Linear DNA sample = 2 x 30ng/ 5microlitre.

ANSWER= 12ng/microlitre

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