Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).
|PLASMID||Vol for 1.5μg (μL)||H2O to 40μL|
Digestion Master Mix
|REAGENT||SINGLE REACTION (μL)||x 5.5 (μL)|
|10x Buffer 3.1 (NEB)||5||27.5|
|TOTAL||50||Add 10μL to each tube|
Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.
Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.
Besides the funky way this gel ran, the digests look to be complete.
Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).