Question

Setup the reaction conditions for EcoR1 enzyme (to digest 1 ug of DNA). Make sure to list all components necessary including the buffers. please label all components you use. 10 pts

Answer 1

1. Add components to a clean tube in the order shown:
        1 µL DNA (concentration 1 µg/µL)
        2 µL 10x buffer
        1 µL restriction enzyme :- ECoRI
        16 µL sterile water
2. Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
3. Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
4. The digested DNA is ready for use in research applications...

Equipment

• Electrophoresis chamber
• Pipetman

Reagents

• Liquid DNA aliquot of your plasmid of interest
• Appropriate restriction enzyme
• Approrpriate restriction digest buffer
• Gel loading dye
• Electrophoresis buffer
• Pipet tips

Procedure

Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours.

If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction. This may involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit, such as a  QIAGEN DNA cleanup kit.

1. Select restriction enzymes to digest your plasmid.

   To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer... Here we will use ECorI as mentioned in question..

2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.

3. In a 1.5mL tube combine the following:

   • DNA
   • Restriction Enzyme(s)
   • Buffer
   • BSA (if recommended by manufacturer)
   • dH2O up to total volume

   The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total reaction volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA to be cut.

   A typical restriction digestion reaction could look like this:
   • 1 µg DNA
   • 1 µL of each Restriction Enzyme
   • 3 µL 10x Buffer
   • 3 µL 10x BSA (if recommended)
   • x µL dH2O (to bring total volume to 30µL)

   

   The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut. By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour. Using this ratio, you can calculate the minimal amount of enzyme for your reaction. However, keep in mind that restriction enzyme activity is determined under ideal conditions with very clean DNA, so using a little more enzyme is advisable. Reactions are often performed with 0.2-0.5 µL of enzyme because it is difficult to pipette less volume than this; 0.2-0.5 µL will likely be more enzyme than you will need, but that's okay because a little more enzyme is usually better.

4. Mix gently by pipetting.
5. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions.
6. To visualize the results of your digest, conduct gel electrophoresis.