Hello everyone, I prepared my vector (pET23b) and got 10.5ug of Vector dissolved in 150ul of nuclease free water and the insert is about 11 ug in 100ul. i wanted to perform a double digestion on all amount of vector and insert but according to NEB the digestion protocol consists
1ug DNA
5ul buffer
1ul enzyme (20000 units/ml)
total reaction volume 50ul
In this case as I have 10ug of DNA the reaction mixture must be
10ug DNA
50ul of buffer
10ul enzyme
total reaction volume 500ul
Which looks me quite strange because I do not have so much enzyme left. Is it possible to perform by any other means? Can you suggest to me a reaction protocol to perform the reaction.
One unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete digest of 1 µg of λDNA in 60 minutes at 37°C in a 50 µl reaction.
Since your enzyme activity is 20000U/ml, in theory, you should be able to digest 20ug DNA with 1ul enzyme in a 50ul reaction in 1 hour.
Your DNA is already in a larger volume so I would just scale up the enzyme and buffer to match your volume i.e
vector 150ul
buffer 17.5ul
enzyme 4ul and 4ul
insert 100ul
buffer 11.7ul
enzyme 3ul and 3ul
Having said that though, if you didn't have enough enzyme, it is still likely to work with 1ul each in the bigger volume. Just incubate longer
Do you tell me how you defined the buffer amount 17.5 ul and 11.7ul respectively
No problem i calculated it myself thank you Amanda...
hope restricition digestion goes well
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