Please show work!!! You are ready to set up your reaction. You are provided with
ID: 208175 • Letter: P
Question
Please show work!!! You are ready to set up your reaction. You are provided with a stock of plasmid DNA that is at a concentration of lmg/ml. You have stock enzyme buffer that is 10X (meaning it is ten-times more concentrated than you need it to be in the actual reaction). You have deionized water (dH20) to use, as needed. As noted above, you want to cut 2ug DNA in a reaction volume of 50ul. To make sure you get complete cutting, you'll use an excess of enzyme so that you use 2.5 units per microgram of DNA (2.5U/ug). The ApeKI enzyme is supplied at 5,000U/ml. How many ul of DNA will you add to your reaction tube? How many ul of 10X buffer will you add to your reaction tube? How many ul of ApeKI will you add to your reaction tube? How many ul of dH20 will you add to your reaction tube? At what temperature will you incubate the reaction?Explanation / Answer
Restriction Digest of plasmid DNA :
Introduction :
- Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences.
- Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction cloning.
- It is also used to quickly check the identity of a plasmid by diagnostic digest.
Reagent :
- Liquid DNA aliquot of your plasmid of interest
- Appropriate restriction enzyme
- Approrpriate restriction digest buffer
- Gel loading dye
- Electrophoresis buffer
- Pipet tips
Procedure :
- Enzyme for cut DNA sequence (ApeKI).
- Determine an appropriate reaction buffer by reading the instructions for your enzyme.
- In a 5,000 U/ml tube combine the following:
The amount of DNA that you cut depends on your application.
Diagnostic digests typically involve 500 ng of DNA, while molecular cloning often requires 1-3 µg of DNA.
- A restriction digestion reaction could look like this:
- 1.6 µg DNA
- 1.6 µL of each Restriction Enzyme ( ApeKI )
- 5 µL 10x Buffer
- 5 µL 10x BSA (if recommended)
- x µL dH2O (to bring total volume to 50µL)
- mix gently and incubate tube at 37oC.