sgRNA purification

I am using the megaclear kit from Ambion to purify my in vitro transcribed sgRNA and the problem is that I get very low yield (8ng/ul) after purification, though the initial IVT yielded ~60 ug! What is the


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Bronze Impalmost 9 years ago

Try using RNeasy mini kit to purify your gRNA.

Silver Dragonalmost 9 years ago

Thanks lot for your quick reply. And I just have another quick question. For the RNeasy mini kit, Should I start from the beginning by adding lysis buffer and chloroform or just diluted my IVT product with elusion buffer and go to the column step directly.

Bronze Impalmost 9 years ago

Follow the exact protocol of RNA clean up in RNeasy mini handbook (page aroun 54-56). Good luck.

Peach Selkiealmost 9 years ago

RNeasy kit says it works for RNAs larger than 200nt, so I wonder if that's the best choice?

Jade Vampirealmost 9 years ago

We use the Post Reaction Clean up Kit from Sigma nowerdays, works fine also for RNA although it says DNA PCR products in the sheet. Or you can use the macherey and nagel kit with the NTC buffer(excludes <50bp). Also there is another macherey and nagel kit for small RNAs which works perfect I used it once from a neighboring lab.

Red Pegasusalmost 9 years ago

I get great yields from the RNEasy kit. Just split the reaction so you don't overload a column.

Orange Trollover 4 years ago

To who used the RNeasy mini kit, in how much water sgRNA should be eluted to maintain the high yield?

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