Hello,
I'm following Thermofisher's "Ion 16S Metagenomics Kit User Guide", and I want to ligate Ion P1 Adaptors, and an Ion Xpress Barcode onto dsDNA amplicons for ion torrent sequencing (p. 16, attached file).
The "Ion Plus Fragment Library Kit" comes with E. coli DNA ligase and buffer. However, I'm may have too little, or the reagents may have suffered too many freeze thaw cycles.
I've heard that both reagents can be substituted with Promega's T4 DNA ligase and buffer (attached file) to yield high-quality results. Both companies' tech support provided inconclusive answers of using T4 with the "Ion 16S...Guide".
Questions:
1. What are your experiences using the T4 reagents for this protocol?
2. What would happen if both DNA ligases and buffers were added to the same library? Would they somehow interfere/inhibit each other's reaction?
Thank you,
Gary
Are you sure that the kit uses E. coli ligase?
I think it's highly unlikely, because E. coli ligase cannot ligate blunt ends, which should be the case after end-repair.
Also, in any aspects, T4 ligase acts better than E. coli ligase. That's why E. coli ligase is hardly used nowadays.
I would assume the kit DOES use T4 ligase. Therefore you may be able to add T4 ligase, as long as you add appropriate amount of the enzyme.
I am unsure, however, if you should add T4 ligase buffer, because we do not know whether ligase in your kit is dead, or reagents (e.g., ATP).
Perhaps you should try by yourself.
I assume you would be able to check if the ligation was successful during amplification of the library, right?
Why don't you try with a small number of test samples?
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