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An animal was anesthetized with isoflurane (VEDCO, St . Joseph MO), and a PTF catheter with a blunted end (Catamount Research, St. Albans, VT) was sutured in place at the bladder dome and tunneled out the abdomen to the nape of the neck where it was then inserted into the end of a 22-gauge angiocath iv catheter.
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Then, the slr1609 gene was excised with NdeI and DraIII (blunted end) from pGQ7 and inserted into the NdeI/ SalI (blunted end) site of pXT68, to generate the plasmid pGQ49.
If you can, buy a set with blunted ends.
The best instrument to use is a small nail scissors, preferably one with blunted ends.
Correct insertion yields a novel NcoI site generated by ligation of the blunted ends, and so NcoI (NEB) linearization of the supercoiled vector was used to determine correct insertion.
The PCR blunted-end product was subcloned into pSTBlue1 acceptor vector, using the Acceptor Vector kit (Novagen, EMD Biosciences, US), digested with kPN1 and SacI and subcloned into kPN1/SacI sites of the reporter vector pGL3 basic.
The ligated products were purified on a MicroSpin S-400 spin column (GE Healthcare), sonicated to obtain fragments with a median length of 500 bp, purified with biotin-streptavidin interaction magnetic beads (Dynal), phosphorylated and blunted-end as above, and ligated to Adaptor-II (sequence available in Additional file 1).
In exercise and dobutamine tests this was almost entirely due to a blunted increase (P < 0.05) in ELVI from rest to peak stress; interestingly, despite a blunted increase in end-systolic elastance index (ELVI) in all dipyridamole tests, still coupling (ELVI/ EaI) reserve was present in normals for a negative arterial elastance (EaI) reserve in this vasodilator test.
To this end, increased cIMT has been demonstrated in adolescents with type 1 diabetes, i.e. in association with a blunted endothelial regenerative capacity and low adiponectin levels [ 11].
"It was like trying to fight with a blunted weapon".
Nor did the cloying fog that had enveloped us in a blunted monochrome world.
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Since I tried Ludwig back in 2017, I have been constantly using it in both editing and translation. Ever since, I suggest it to my translators at ProSciEditing.

Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com