the protocol said
(Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture)
My plasmid concentration is 114 ng/µl
The cell mixture ( competent cells ) is 50 microliter
I used M*V = M2*V2 but it gave higher number
Dear Ryan .. ,
At first, you can dilute your plasmid dna or continue with your 114ng/microliter concentration.For example , if you want to add at 50 ng/ microliter plasmid DNA, you must follow ;
M1.V1=M2.V2
114 . x microlitrer = 50. 1 microliter (it is for the competent stock)
x microliter = 50/114= 0,440 microliter.
Add 0.440 microliter from your 114 ng/microliter plasmid dna to the competent cells. However, because this amount is too low, you can dilute your stock to the 50 or 100 ng/microliter. If you have 5 microliter 114ng/microliter Plasmid and you want to end with 50ng/microliter concentration ;
M1.V1=M2.V2
114.5= 50.x => x = 11,4 microliter
add 6,4 microliter of water to the 5 microliter of 114ng/microliter Plasmid solution.
From this solution, you can take 0,5 microliter to your competent cells to handle 50 ng/microlite plasmid concentration at the end.
Hi Ryan ..
Dilute your plasmid DNA in TE buffer @ 1: 2 that will make the new concentration 57 ng/µl (114/2=57). As your protocol says adding 1pg-100ng, so you can add 1 µl of the diluted plasmid DNA (containing 57 ng) into 50 µl of the competent cells.
The protocol gives you a fairly large range of the amount of DNA to add to your competent cells. If you use given lower and upper limits, add anywhere from 1 pg - 500 ng of DNA. So the amount depends on how much you have, how efficient your ligation may be (if this is cloning). If you are just transfecting a pure plasmid into E. coli, then any small amount will do.
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