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 WEcoRI digest A. Information for calculations: The total volume of each reaction will be 65 ul Each reaction needs to include 1x buffer E (supplied as a 10x stock solution)o 5 The pUC reaction needs 2 ?g DNA (stock solution is 1 mg/ml) The ? reaction needs 15 ?g DNA (stock solution is 500 ?g/ml) Each reaction needs 15 units of EcoRI (stock is 5 units/ul) Balance the volume of each reaction with sterile water 33.33 Perform the necessary calculations as part of the pre-lab assignment. During lab when you get your pre-lab handed back, if your calculations were correct, record them in your lab notebook and proceed with the protocol. if you need to redo your calculations, check them with me before proceeding.

Explanation / Answer

Total reaction volume = 65 uL

10 X buffer = 6.5 uL

pUC DNA stock = 1 mg/mL

Required amount = 2 ug

1 mL ---> 1 mg

2 X 10^-3 mg ----> ? mL

= 2 uL

Lambda DNA stock = 500 ug/mL

Required amount = 15 ug

1 mL ---> 500 ug

15 ug ----> ? mL

= 30 uL

Eco RI = 3 uL

pUC reaction:

DNA = 2 uL

Buffer = 6.5 uL

Enzyme = 3 uL

Water = 53.5 uL

Lambda reaction:

DNA = 30 uL

Buffer = 6.5 uL

Enzyme = 3 uL

Water = 25.5 uL

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