Greetings esteemed researchers!
I am relatively new to the field and would greatly appreciate some guidance on a challenge I'm encountering in my research.
We're currently engaged in a process akin to cloning, involving Hifi assembly to create linear DNA. The workflow entails N core extension, N core amplification, gene amplification and purification, C frag amplification and purification, Hifi assembly, and final expression and purification.
While following a protocol initially designed for cloning, we've observed that the expression of one particular gene isn't meeting our expectations. Upon consultation, it was suggested that we optimize the Hifi assembly step. (https://www.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)
However, given the unique nature of our experiment, I'm unsure where to begin in terms of adjusting the ratios.
Our fragments have varying concentrations, and the molar ratio used for assembly (insert:vector = 2:1) might not be optimal for our setup. Could anyone provide insights or advice on how to approach optimizing these ratios effectively?
Your expertise and assistance in navigating this challenge would be immensely valuable to our research efforts. Thank you in advance for your support and guidance.
Ps. As I'm relatively new to this field, I find it challenging to discern the fragments clearly or to grasp the insights that come with experience. any help is appreciated!
Hi,
How long are the fragments you are trying to join? And how many are there?
Thank you for your response! Bruno Salomone Gonzalez de Castejon
1. 3 fragments
Ncore - Gene - C frag
2. length in bp
Ncore - 100 bp
Gene - 1503 bp
Cfrag - 230 bp
Thanks in advance
No worries! So have you PCR'd out these fragments from a genome, or have you bought them as synthesised DNA strings or shuttled in a plasmid vector?
To perform HiFi you need homologous regions between the fragments, how long are these?
Also if these are your final fragment sizes after amplification and purification, perhaps there are other methods to join them that could be easier. Such as overlapping PCR, I got a good protocol that I made if you need it.
Thank you for the response!
All 3 fragments are PCR products and purified.
Ncore - 100 bp but with the primer the total length of Nfrag is 184 (after PCR)
Gene - 1479 bp but with primer the total length is 1503bp (after PCR)
Cfrag - 230 bp (after PCR)
all have different concentrations, so optimizing the ratio can be important but i dont know how to begin with.
Im getting a smear band (pic attached) in the final expression PCR after HiFi assembly (using that as template).
It's a bit tricky as it's new to me. Im confused 1) if the Hifi assembly should have a minimum DNA concentration? 2) should the gene concentration should be more than Cfrag and N frag etc.
Any comments or suggestions will be appreciated!
I would like to get the protocol that you have, and thank you for offering!
Email id: [email protected]
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