Protocol: I. Setting up your restriction digests: each team will set up a pUC19/

Question

Protocol: I. Setting up your restriction digests: each team will set up a pUC19/EcoRI digest and a MEcoRI digest. A. Information for calculations: The total volume of each reaction will be 65 Each reaction needs to include buffer E (supplied as a 10x stock solution) The puc reaction needs 2 DNA (stock solution is 1 mg/ml) The reaction needs 1S DNA (stock solution is SO/ml) Each reaction needs 15 units of EcoRI (stock is S units/ul Balance the volume of each reaction with sterile water 1. If you have a DNA solution that is labeled as 1 mg/ml, how much DNA is in 1 ? 2, what volume of this DNA would you use to get 2 g of DNA? 3. If your DNA solution is 500 g/ml, what volume would you use to get 30 ? 4. If you have an enzyme with a concentration of 10,000 units/ml what volume would give you 10 units? 5. If you set up reactions with 10 units in each reaction, and plan a total of 16 reactions, what volume of this enzyme would you need? 6. Perform the necessary calculations to set up each reaction for Lab 3. Use the information provided in part A of the protocol

Explanation / Answer

1. l mg - 1000 ug. 1 ml - 1000 ul.
so in micro units the 1mg/ml = 1000/1000 ul, which means the final concentration is 1ug/1ml. so, 1 ug of DNA is present in 1 ul of DNA.

2. For 2ug of DNA, 2ul of volume is enough from the above stock.

3. 500 > 1000 ul
30 > ?

= 30 x 1000 / 500 = 60. The 30 ug of DNA would present in 60 ul of volume.

4. 10000 > 1000 ul
10 > ?

= 10 x 1000 / 10000 = 1. The 10 units of enzyme would present in 1 ul of volume.

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