Greetings! Here is tuesday now..
After coujugation of amine coupled oligonucleotide to PEG particle, we hybridized and it seemed like fluorescence this point, possibly DNA also overall negative charge and COOH of particle also negative charge wish to streptavidin first conjugation Then we proceeded the hybridization after biotinylated DNA was reacted.
However, there were two major problems. Firstly, despite that, the fluorescence appeared as if it drew only a frame with a green highlighter as if it were just fluorescent (only the frame of the fluorescent particle was seen), the second is incorrect in the middle of the reaction It seems that all the particles have improved.
streptavidin conjugation, biotinylated DNA reaction and hybridization progressed for 2 hours each for a total of 6 hours. Hybridization, which took place in 4 hours, has progressed for a total of 8 hours. (2 h + 2 h + 2 h / 2 h + 2 h + 4 h)
However, there is one pinch point, but it means that it proceeded while vortexing perfectly during the reaction time. When amine modified oligonucleotide was immediately conjugated, it was vortexed for 2 hours in conjugation of oligonucleotide and 2 hours in hybridization for a total of 4 hours. And in this experiment, I vortexed a total of 6 hours and vortexed a total of 8 hours.
It reacted for 6 hours in total, the fact that the particle that occurred indiscriminately had quite a few jokugin, and reacting for 8 hours in total was a broken state with all the particles.
Is there a possibility that PEG particles will break if vortexing for a long time?
PS: I will try to advance the reaction while shacking broken because of vortexing that has progressed for a long time, but it does not mix well with a very small amount. (There is no rotator)
It is better to perform the reaction in a shaker platform instead of using voretex for mixing biologicals.
@Alemu Tekewe thank you for your answer..
BTW, my reaction volume is low(under 100ul, maximum 230ul) so I didn't using shaker before. (now I use shaker)
Physical stability testing is conducted by subjecting the biological preparations to a horizontal shake test (100 strokes/min at 37C) to cause accelerated decomposition.
I'm not sure I understand your methodological reasoning, streptavidin-biotin conjugation reaches equilibrium in ~60 seconds in ~1 mPa * s viscosity solutions, why proceed for 2 hours? I'm also not a huge fan of NHS ester conjugation, if that's what you're using, you might want to consider switching to soft conjugation reagents such as SuFEx click chemistry.
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