Page of 3 -ZOOM+ 2. The following table is a partial protocol for a double diges

Question

Page of 3 -ZOOM+ 2. The following table is a partial protocol for a double digest that must be done on a DNA plasmid. Please fill out the missing information in the table to successfully complete your experiment. volume of your reaction needs to be 50 L. Show your calculations below (12 points). For the restriction enzyme Notl the unit U/uL means Units of enzyme per ul. A Unit of an enzyme is usually the amount of an enzyme required to convert 1 mol of substrate to product per minute. The final Final Concentration or Amount Stock Volume Needed Concentration 10X 29 ng/ Reaction Buffer Plasmid Notl Xhol BSA 10 U/L 70 HM 0.05 mg/mL 100 ng/mL MQH20 3. Your TA is going to have to make a lot of 0.5X TAE for the amount of agarose gels we are going to be running. 0.5X TAE consists of 0.02 M Tris-base, 9 mM acetic acid and 500 M EDTA. To make her job easier she will be preparing a 10x solution of TAE and diluting it to 0.5X TAE. Could you please tell her the amount, in grams, of each component she must add prior to adding water to reach one liter to make the 10x solution? Also, what fold-dilution must she make to go from 10X to 0.5X (8 points)?

Explanation / Answer

Answer:

Part 1:

Based on the given data, below is the table with all calculations:

Calculations used:

Plasmid:

XhoI Restriction enzyme

BSA:

Milli-Q water:

Required volume = 50ul- (34.5 + 1 + 0.1 + 5 + 1) = 8.4 ul

Part 2:

Molecular weight of different compounds used in the buffer preparation are:

0.5X TAE consists of

10X Solution will contain

So add, 48.4 g Tris + 10.8 g Acetic acid + 2.9g EDTA to total 1 L (1000ml) water.

Fold dilution from 10x to 0.5x = 10/0.5 = 20x = 20 fold

Therefore a 20 fold dilution is required to prepare 0.5x buffer from 10x stock (50ml 10x Stock + 950 ml MQ Water)

Stock Concentration Final Concentration or Amount Volume Needed Reaction Buffer 10X 1 X 5 ul Plasmid 29 ng/ul 1 ug 34.5 ul NotI 10 U/ul 1 ul XhoI 70 uM 1 uM 0.71ul (1ul conc. = 1.4 uM) BSA 0.05 mg/ml 100ng/ml 0.1 ul MQH2O 8.4ul Total Reaction Volume 50ul

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