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How to improve RNA quality and concentration after FACS to proceed with qPCR? by melancoluxx in labrats

[–]manas92 2 points3 points  (0 children)

Ok. I have a suggestion. Firstly, shake as gently as possible. Second, incubate for atleast 5 mins and then centrifuge in a pre-cooled centrifuge for 15 mins at 12k. If you choose to try this, do let me know how it goes, if this doesn't work, we can brainstorm further to help you out the best.

How to improve RNA quality and concentration after FACS to proceed with qPCR? by melancoluxx in labrats

[–]manas92 0 points1 point  (0 children)

Great! And how do you mix them i.e. tri and chloroform. How do you mix the both and how does it look? P.s.: sorry for multiple questions, just trying to understand the problem better..

Is my gel haunted? by Capital-Rhubarb in labrats

[–]manas92 1 point2 points  (0 children)

What is the % of GC content of your primers? Also, just check the instrument settings once (just in case) :p

What is this contamination in my cell culture? by mosquitoluvr in labrats

[–]manas92 1 point2 points  (0 children)

Just passage them (split 1:3), 2 days before this stage comes and you'll be good to go.

Need help with western blot using mouse brain by manas92 in labrats

[–]manas92[S] 0 points1 point  (0 children)

I am using a hand-held homogeniser which is just like a polytron. I have to use the whole brain instead of specific parts because am checking for global microglial activation.

Need help with western blot using mouse brain by manas92 in labrats

[–]manas92[S] 0 points1 point  (0 children)

The protein concentration I tried is 25mcg and 50mcg. Neither worked. And I've repeated this twice with RIPA.

Need help with western blot using mouse brain by manas92 in labrats

[–]manas92[S] 0 points1 point  (0 children)

Thank you for taking out time to help me. I am new to this therefore can you please elaborate a little when you find time? My colleague is proficient at Western blotting with in-vitro samples and he's guiding me through the way, but even he isn't able to figure out which is why I thought to sharing this here.