Hi, I have recently started my cloning experments and don't have much experience in doing ligation reactions. I have five insert parts with each having 10ng/ul concentration and i need 200ng of inserts in my final 20ul PCR reactions with 50ng of plasmid. But with this requirement the need of inserts itself will be around 20ul which is not making sense to me.
Therefore, to achieve these reactions, what adjustments do adjustments toadjustmentstoI have to consider for proper calculations, do i need make any adjustment with total volume of reaction or something else. Please guide me with this some explanation if possible as i am a bit confused with this situation.
Thank you in advance.
How did you get the 200ng figure? Generally you aim for a 3-5:1 insert: vector molar ratio and since the inserts tend to be smaller than the vector it mean you need less insert, in ng, to get the correct molar ratio.
If you actually need these amounts than you can either try to concentrate the inserts or scale the amount of vector down so you can use less insert while maintaining the ratio.
The optimal molar concentration of DNA insert and plasmid for ligation is contingent upon the chosen ratio of insert to vector, typically set at 3:1 or 5:1 (insert:vector). This determination is influenced by both the size and concentration of the insert and plasmid vector. For convenient and accurate calculations, the NEB ligation calculator is recommended. A link to the calculator is provided for your convenience, facilitating the process.
https://nebiocalculator.neb.com/#!/ligation
If you make a standard ligation with T7-ligase, sure you have to calculate molar ratio, 3:1 or 5:1 (insert:vector) is OK for large inserts, 500-1000 nt, for small ones we used a ratio up to 10:1.
For the Gibson reation this is better to keep the ratio 1:1.
I got confused between the ratio of inserts and vector, which has been resolved now after your clarifications. Thank you so much, Yoav Lubelsky Airat R. Kayumov Usha Kantiwal
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