BACKGROUND:
I'm writing my undergrad thesis on LAMP and RT-LAMP reaction optimization LAMP has never been used at my college so I'm establishing a protocol for future student use and a premises for downstream application. I'm new at LAMP, but I seem to have pulled off a successful amplification of the 6-phosphofructo kinase from a plasmid (pCR Blunt-II TOPO) isolated from E. Coli on my first try, and got the familiar banding pattern with several of my samples. Since I'm establishing protocol for future student use, I'm interested in the downstream applications of LAMP amplification products, specifically, whether LAMP might be used in stead of PCR to synthesize labelled DNA probes for blotting.
QUESTION:
Whether or not I can combine a restriction digest, followed by a denaturing step, to yield one single stranded product, visualized in a denaturing agarose (or acrylamide) gel.
REACTION DETAILS:
My highest yield was 900 ng/uL (66.9 C, 8mM Mg2+) from 72 pg/uL of template (the last lane in the image, lane 14). I used NEB Bst 3.0 polymerase at three different temperatures (66.9, 65.0, and 63.3o C) and four concentrations of Mg2+ (2, 4, 6, 8mM) for 60 minutes before heat-killing the enzyme at 80 C for 10 minutes (Bio-Rad T100 thermal cycler). Negative control showed no amplification. I ran the products out on a 0.8% agarose gel and visualized them with EtBr. cDNA concentrations were measured using a Qubit 3.0 fluorometer.
RT-LAMP reactions are in the incubator right now, results to follow!
Thanks!
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