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Author: Subject: mir302/367 synthesis for IPSC generation
SupaVillain
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[*] posted on 18-5-2015 at 14:40
mir302/367 synthesis for IPSC generation


Biotechnology is one of the most annoying things to try to learn via internet. Everything is so disorganized and nothing seems to be straightforward.

What Im trying to learn is how to make micro RNA's. Specifically the mir302/367 cluster. Along with valproic acid they induce pluripotency in humans

There is a plasmid vector pLOVE mir302/367 product from addgene however I think this is something totally different than "actual" mir302/367. Never heard anything about plasmid vectors involved in inducing pluripotency. Very confused here.




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SupaVillain
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[*] posted on 20-5-2015 at 17:21


List of things I think I need, some things I may be wrong about...

-RNA Polymerase II for transcribing the hairpin microRna
-Droscha DGRC8 Pasha. I think DGRC8 is a synonym of Pasha, pasha is pretty much paired alongside Drosha. This brings it through stages like... i think to priRna and preRna and then mature microRna
-Exportin 5/XPO5 used for exporting out of nucleus into cytoplasm
-Dicer
-Ran/ GTP binding nuclear protein

Learning Bio as i go seems like ill have to figure out how to get all these genes transcribed onto proteins or etc. And then they can be applied.




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Bonee
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[*] posted on 25-5-2015 at 13:41


My greatest problem with biotechnology is that you need expensive equipment, although there has been some advancements on the diy field.

The problem with microRNAs is that most protocols are not for isolating it but
for making a virus that can transfect cells to express the microRNA as in the addgene vector. Because you need a constant expression of these microRNAs for pluripotency they can't just be added to the cells...

[Edited on 25-5-2015 by Bonee]
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SupaVillain
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[*] posted on 5-7-2015 at 07:25


Im not so sure about that, i believe that the said micro rna's will induce pluripotentcy as long as valproic acid is added as well. Theres a lot of DIY bio stuff out there, ive got parts for a lightbulb PCR, like 25 or 30 bucks, couldve made a gel electrophoresis box for cheap but bought an industrial one off ebay for 40 bucks, still need to get the gel and staining chemical, as well as all the PCR stuff, ill have to make an oligonucleotide synthesizer so that i can make the primers instead of buying them. Its not TOO expensive it's really just the initial costs of the main machines. Still need a UV light, filter, and maybe even camera for the gel box too



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SupaVillain
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[*] posted on 5-7-2015 at 07:32


See here,

medcraveonline.com/JHVRV/JHVRV-02-00044.pdf

They get expressed as proteins, the mir302 367 cluster does i mean, however very inefficient without VPA




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