I'm running in vitro transcription with Maxiscript T7 polymerase, to synthesize mRNA from my genes of interest from plasmid. running them on 10% denaturing acrylamide gel for purification to cut the dark sports, according X-ray imaging and incubate in gel elution buffer (0.5M NH4AC, 1mM EDTA, 0.1%. SDS).
On the next day, i'm spinning my sample, to aspire supernatant and mixing them with 1/10 V. 3M NaAC, 10 µg tRNA and 2 µl glycoblue (per sample) . 45 min incubation under -20 and there is no trace of radiation in the alcohol but the pellet is "hot".
After that, I'm mixing my hot-label and target RNA samples for the hybridization, again precipitating with the salt, glycogen and ethanol and dissolve the pellet into hybridization buffer (80% formamide, 2M NA citrate, 3M NaAC pH 7 , 0.5M EDTA and 0.5g PEG).
After the overnight hybridization, I 'm digesting single stranded not hybridized RNA, then making Phenol extraction and again precipitate the last time, prior running on the gel.
The problem emerging after third precipitation of hybridized samples. the ethanol has radiation trace and out of i changed time and temperature (from 45 min to 3 h) ( from -20 to -70) for the precipitation, there are still radiation trace on it. I'm looking forward for your advices how to maintain hot status for my pellet prior dissolving into Gel loading Buffer II.
If I understand correctly you're doing some kind of Northern blot, you radio-label your probe, which is hot and you hybridize it. But we don't know about your results from the Hybridization step. Did it work? Did you see a band?
I guess what you're attempting to do in the last precipitation is to recycle the probe for another round of hybridization and you're are detecting loss of specific activity in your pellet probably due to degradation during hybridization. There's not much to suggest except to accept the loss of specific activity in the sup.
Unless I'm missing something, this is what I gather from your description.
I see the bends, but the hybridized sample with 400K cpma label has the almost same intensity as 2000 cpma positive. fresh label control. I have this problem for last couple of blot, previously it was fine. No hot trace observed in ethanol after precipitation before.
Highly energetic isotopes like 32-P decay fast and thus the specific activity halves in 14.3 days.
Exposure to X-ray film sometimes gets saturated so there's a plateau of detection, so the difference in intensity between the 2K cpms probe and the 400K cpms might be due to decay between experiments ( I don't know how many days passed between one experiment and the other).
Assuming you prepared both at the same time, that difference could be due to lack of linearity in the plateau phase.
Radiolabeled nucleotides "could" exhibit decreased chemical stability and radioactivity is released in the sup, doesn't incorporate into the hybridization probe. Those are my thoughts now.
When you get fresh isotope there's high specific activity but it decreases with decay.
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