I have a set of a couple dozen DNA constructs that I plan to order for nucleosome reconstitutions, I was wondering what are the pros and cons of having them as fragments vs cloned. I don't like having the cloned version because they're significantly more expensive (about >2x vs fragments). I think it's good practice to have them cloned eventually for long term storage but haven't had direct experience with this.
I don't intend to express these sequences, rather, I'd simply like to PCR them in mass quantity.
I would appreciate your thoughts on this.
Thanks
it depends on how accurately you want to the sequence to be. Fragments are cheaper but not sequence verified on the other hand cloned construct are sequence verified. What you could do is sequence a portion of the PCR reaction coming out of the fragment to make sure that the original template is correct (of course keep in mind that the nucleotides that are the results of PCR error are not going to be clear from the seq reaction as the majority of copies with have the correct sequence but it give an indication whether the original template is mutated or not).
it depends on how accurately you want to the sequence to be. Fragments are cheaper but not sequence verified on the other hand cloned construct are sequence verified. What you could do is sequence a portion of the PCR reaction coming out of the fragment to make sure that the original template is correct (of course keep in mind that the nucleotides that are the results of PCR error are not going to be clear from the seq reaction as the majority of copies with have the correct sequence but it give an indication whether the original template is mutated or not).
Dear Tuan,
I recommend Hanna's suggestion. However, practically speaking, it would be very difficult to manage so many constructs without cloning them. If you intend to store them for really long periods, I would suggest you to proceed with cloning them. Considering the amount of time and manpower you would invest in locating a misplaced construct (if stored as fragment), I would modestly recommend you to proceed with cloning, no matter how expensive it is. I, though being an independent researcher (no great funding or sponsors), do this.
Another point I will glad to above answer is using of polymerase having proof reading.
Regards
Dear Tuan,
I would suggest to order the fragments cloned which are sequence-verified. Sequence-verification is an important point as Hanna pointed out. You can transform the plasmids into E. coli and amplify the fragments anytime you need to. You will save money long term. Also, you may want to add to the order a plasmid DNA prep (like a 50µg prep) for a small additional cost. The DNA should be enough for your large scale PCR amplification for a long while. Actually, we do that for the past many years for DNA fragments we used for our nucleosome reconstitutions.
Best wishes,
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