Question

1. to measure DNA concentration, if you take 1/10 of a sample and read the A260 and the reading is 1.5. what is the origin Dna concentration?

2. how to prepare a 200 ml 12% 29:1 polyacrylamide gel?

Answer 1

1) DNA concentration= A260 reading/ Path length * Dilution factor* 50 microgram/ml

Path length of cuvette is usually 1 cm

A260 reading= 1.5

The sample taken was 1/10^th. However, no dilution was performed on the sample. Hence, dilution factor is 1.

DNA concentration of sample taken= 1.5/1cm * 1* 50 microgram/ml= 75 microgram/ml

DNA yield (microgram)= DNA concentration * total sample volume

If 1 ml of volume fits in the cuvette for absorbance readings, then

If 1/10^th of the sample was used, total sample would have 10 ml volume

In this scenario:

DNA yield in original sample= 75 microgram * 10= 750 microgram

The calculations can be done accordingly.

2) 29: 1 Acrylamide can be prepared as a 30% solution or 40% solution of Acrylamide.

The below preparation is for 30% solution.

Preparation: 29 g acrylamide

1g of N,N'-methylbisacrylamide

Add 60ml of water. Adjust the total volume to 100 ml with water.

Filter the solution through 0.45 micron pore size filter and store in fridge protected from light.

To prepare 10 ml of 12% polyacrylamide resolving gel,

30% Acryamide (29:1)= 4 ml

1.5 M Tris buffer pH 8.8= 2.5 ml

10% SDS= 0.1 ml

Distilled Water= 3.3 ml

10% APS= 100 microliter

TEMED= 10 microliter

10% APS and TEMED are added last, just prior to pouring of mixture to the plate.

To prepare 200 ml of 12% 29: 1 Polyacryamide gel

30% Acryamide (29:1)= 4*20= 80 ml

1.5 M Tris buffer pH 8.8= 2.5 ml *20= 50 ml

10% SDS= 0.1 ml *20= 2ml

Distilled Water= 3.3 ml *20= 66 ml

10% APS (Ammonium persulfate)= 100 microliter *20= 2000 microliter= 2ml

TEMED (N,N,N',N'-tetramethylethylene Diamine) = 10 microliter *20= 200 microliter= 0.2 ml

Mix all ingredients except 10% APS and TEMED first.

10% APS and TEMED are added last, just prior to pouring of mixture to the plate.