can anyone please explain picomole calculation in gibson assembly like what is vector and insert ratio to be added in reaction and how to add a picomole to a reaction?
NEB recommends 50-100 ng vector and a 2-3 fold excess of insert.
You usually know the length of your vector and of your insert. Then you calculate or measure the amount of DNA in µg. The MW of a base pair is known (app. 660 daltons). Now you can start to calculate the pMol/µl of your DNAs. Example: In case of a 1kb fragment 0.66µg are 1pMol.
See the formulas in e.g. in the Roche "Lab FAQs"
https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/12115972001_10.11.pdf
or the calculation in the NEB ManualE5510 calculating with 650 daltons:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
50 ng of 5000 bp dsDNA is about 0.015 pmols.
50 ng of 500 bp dsDNA is about 0.15 pmols.
In the NEB biotools website there is a calculator to transform, ng to pmol, is what I used and works, then you only need to set the proportion 1/2 or 1/1/1...
https://www.neb.com/tools-and-resources/interactive-tools
http://nebiocalculator.neb.com/#!/
50 ng of 5000 bp dsDNA is 0,01618 pmols, 9,75x109 Copies...
50 ng of 500 bp dsDNA is 0,1618 pmols, 9,75x1010 Copies...
hii joy..thankyou for reply sent u a mesege to inbox can u check it...waiting fr reply
Hi,
I have some trouble getting my fragments assembled. I put 100 ng of my vector backbone 9947 bp and 300 ng of my fragment 5650 bp, I incubated for 1 hour but still 2 fragments on agarose gel. Can you please help me?
For a 20 ul reaction, I use 30 ng of vector (they are usually 6-7 kb). While I do use the above mentioned formula, I find that adding more insert makes the reaction far more efficient.
3kb insert: 100-150 ng
1 kb insert: 50 ng
smaller than 1 kb insert: 25 ng
Than incubate at 50C for 1hour in Gibson mix.
The competent cells are also a variable to watch out for. My constructs never gave colonies when transformed onto DH5alpha cells, but worked wonderfully with Stellar cells.
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