I studied the effect of the crowding agent on recombinase polymerase amplification using the TwistDX kit. I prepared the rehydration buffer in house following the company's patent table and noticed that removing PEG from this buffer gave no amplification. I tried other crowding agents such as PVP and Ficoll and they worked equally well.

I am now studying different isothermal amplifications based on the strand displacement method, using LAMP and Nicking ensìdonuclease-mediated strand displacement amplification.

Is the presence of crowding agents so important in this type of amplification?

Is it better to use Bst2.0 or Bst3.0 (from NEB)?

What is the final concentration of Mg++?

What is the final concentration of primers?

Thanks :)

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