February 26, 2006 at 7:18 pm #3789SepalsParticipant
Hi I’m stuck on part of some cw I have to do. I was wondering if any1 could give me any advise please as I can’t seem to work it out on my own.
This is the info and the questions I’m stuck on are below. Thx. 🙂
1. In order to be able to use the Gyrase enzyme in the supercoiling assay, the enzyme needs to be diluted. At present it is supplied as 5U/µl in 50% (w/v) glycerol. The glycerol is used to keep the enzyme stable for long term storage but at this concentration it inhibits the Supercoiling Assay. Therefore we need to dilute the enzyme by adding 18µl of Dilution buffer to the tube containing 2µl of DNA gyrase
Question: After the dilution step how many units of enzyme do you have per µl?
Question: After the dilution step what is the percentage of glycerol (w/v) in the plasmid DNA solution? (Show calculation)
February 26, 2006 at 9:50 pm #41865sdekivitParticipantquote Sepals:
When the first question has been answered, the second one should be easy, so i’ll help you with the first one:
we have 2 uL of a 5U/uL DNA-gyrase-solution in 50% glycerol stock. Now we add 18 uL of dilution buffer, so the total volume has become 20 uL
–> this means that the dilution factor is 10 times, so in the diluted solution we have 0,5 U / uL DNA-gyrase.
(Second method: 5 U/uL –> we have 2 uL, so that means that 2 uL contains 10 units DNA-gyrase.
The total volume became 20 uL, thus 10 units / 20 uL = 0,5 U/uL DNA-gyrase)
February 27, 2006 at 10:32 pm #41932SepalsParticipantquote sdekivit:
Thx a lot for offering to help. I contected a friend who helped me and I used the second method.
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