How to work and handle compounds under argon?

These are universal primers for 16S rRNA amplification;

F24 (5’- GAG TTT GAT YMT GGC TCA G -3’)

Y36 (5’-GAA GGA GGT GWT CCA DCC-3')

What are the representative pairs for YMWD?

How to find the relative density of the 3D printed cellular structures like honeycomb, double arrowhead?

I am using a pET28B vector to clone my 2.1kb insert digested with BamH1 (fwd) and Xho1 (rev) restriction enzymes. I'm using over 1ug of DNA for restriction digestion, kept at 37°C for over 3 hours. For cohesive end ligation, I'm using Promega T4 ligase, and keeping the setup (3:1, 5:1 and 7:1; insert: vector) for 16 hours before transforming in Dh5alpha competent cells followed by plating on Kan plates. I've been doing this for quite sometime now. But everytime, all I get are false colonies. I've tried to troubleshoot every single step but to no avail. My ligase is also new.

I have run these westerns a billion times and something or the other doesn’t work out. For eg, my gel ran straight and fine but when I transfer the gel to my membrane, half of the membrane shows no protein and the other half shows this weird curvy transfer. For context, I ran precast gel 4-12% transferred to membrane using iBLOT P0 dry transfer. Then, I cut the membrane into half (the two halves being biological replicates). One half shows barely anything and the other membrane is curved. What am I doing wrong? Samples are lyses in SDS licor loading buffer

stacking gel 5%,running gel 8%

first band includes catalase(400KDa） and glucose oxidase（160KDa) , small molecular crosslinker(MW<1000) is used ,but no aggregates showed in this band(should have been above than most other bands),why the band below showed like dumbbell-shaped?

for other bands, is the concentration of protein too high because the color of bands is deep and bands are long? why it showed a funnelform?

could any beautiful people help me out of this, I would be so appreciated of your kind answers. your advice is of great help for me as a beginner. Thanks for help!

I am getting smear on gel after using linear DNA (amplicon) as a template while this is not the case with circular DNA (plasmid) as a template with same set of primers. I have used various amounts of template, gradient PCR, lowered the amount of polymerase; but, no success. I am using Phusion™ High-Fidelity DNA Polymerase Kit from Thermo. I will highly appreciate your input.

How does PCR conditions affect specific target ?