u/adventuriser

I have a gift card, and want to get some fly tying materials. What materials are worth it from there? They have a lot of brands i haven't seen in this sub, and the costs are generally a little higher than dedicated fly tying shops

Looking to host visitors around that time next year, and i assume the hotels are crazy expensive then?

SOS. I'm struggling with really low signal northern blots, even for mRNA that should be highly abundant.

I'm wondering if my probe labeling or concentration is the problem. To label my DNA oligonucleotide probe, I follow the following recipe:

• 1.5 µL 10 mM oligo stock
• 5 µL H2O
• 1 µL 10X PNK buffer
• 1 µL T4 PNK (10 Units/µL)
• 1.5 µL of 32P-ATP (specifically this one) (3000Ci/mmol 5mCi/ml)
• Incubate at 37 degC, 45 min, clean by G50 microspin column

By rough estimate with Geiger counter, my labeling efficiency is like 75% which seems about right (75% of radioactivity is contained within probe, 25% remaining on the spin column).

I then add the entire probe to 10 mL of ULTRAHyb-oligo hybridization buffer and hybridize overnight. (Final probe concentration in the hybridization buffer is therefore 1.5 µM)

Is my probe:ATP ratio off in the end-labeling reaction?? Like maybe I have an abundance of unlabeled/cold probe that would effectively block the signal from the hot probe??