Cell and Molecular Biology Lab

[Lukas!]

My professor hasn't been very helpful and I need some help with this.

 

You wish to perform a restriction digest in a 20 microliter volume. Your DNA is .60 micrograms/microliter and you wish to digest 6.0 micrograms. Your restriction enzyme is provided as 2U/microliter. What are the appropriate volumes of

Water:

10x buffer:

DNA:

Restriction enzyme:

I saw one of the professors watching movies on his computer during his office hours and he said, "read the lab manual" when I asked him for help. He wrote the lab manual, and did a pretty poor job. Can anybody explain to me how to solve this problem? First off, what is the activity? (I think it's the "U" but I don't know what that means). What is the component "buffer"? What would really help me is the equations without the numbers and an explanation of why I'm making the calculations step by step. I really hope someone can help me.

[CharonY]

Here some primers: U is indeed the activity (short for unit). You need to figure out/decide how much enzyme activity (how many Us in total) you need in your assay.

 

With that known, you got the volume of your enzyme. Next DNA, here you got all the info. How many microliters of your DNA solution correspond to 6 micrograms?

Then there is the buffer. 10x refers to 10 times concentrated. What concentration, should be in an assay (hint: it may depend on the enzyme)?

 

With these quantities known you only need to fill up with water to the final 20 microliters.

You may also want to check out the websites of restriction enzyme manufacturers, such as Fermentas or NEB.

 

 

[Lukas!]

Thanks! I really appreciate this.