I am looking for strategies which most of the commercial companies use for production of 1 Kb DNA ladder.
I did come across good journal for a 100 bp ladder where they have designed primers using pUC57 and pET 21 vectors and were successful.
From my understanding, designing primers for a plasmid is good till 2000-3000 bp, but what if one wants to design a 1 Kb ladder (which has bands of larger amplicons)
Seeking any patents/strategies used by laboratories around.
Thank You.
piece of cake))
just take plasmid or cosmid and insert 2 unique restriction sites with the desired length in between
1 kb = 1000bp. Plasmids can be used to make ladder of up to 3000bp or more, but for a ladder you will need fragments of many different and known sizes, which will consume a lot of time to standardize! You could also use CRISPR to generate fragments with specific sizes, but that would be expansive.
It's just a restriction digest of a known piece of DNA. Make a plasmid with concatenated pieces, digest and there you go. The hard part is getting the bands a uniform brightness.
Hey all. I do have a strategy for 100 bp ladder (Ranging 100-2000 bp with increment of 100 bp) using PCR amplification of pUC57. Please find the reference if one is interested in making their own 100 bp ladder in lab
But for a 1 kb ladder ( ranging 500 bp - 10 Kb) one has to get restricted fragments of more than 2 kb or PCR amplified products of more than 2 kb. Now to my knowledge we can use cosmids as mentioned by Andrey.
Katie, when we use restriction digestion yes there will be an issue of getting uniform band intensity. THats the reason I would prefer to go behind PCR based amplification of bands and then gel purify; quantify and put into eqvi molar. Thus I am looking for any standard methods in PCR and using ligation of monomers to make larger fragments.
Please share your inputs to further discuss regarding this .
THank You.
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