Transcribing gRNA by mMessage mMachine T7 instead of MAXIscript T7 kit

[agnik dasgupta]

Hi,

So I have been using mMessage mMachine T7 kit to make the guide RNA from DR274 plasmid after digesting it with Dra1. Having trouble to get RNA. Can anyone suggest whether I should use MAXIscript instead of the mMessage. 

thanks

agnik

[Mike Peters]

i'm about to do the same thing. anyone know which is better?

[jb]

I think most use the Maxiscript kit for sgRNA IVT. It helps to increase the template concentration. We usually use ~300-500 ng per 20 ul reaction volume if the template is gel-purified DNA (DraI digest yields a small fragment containing T7 sequence, target and guide RNA). If you don't gel-purify but precipitate the whole digest, I would increase the DNA template amount to more than 2 ug for a 20 ul IVT reaction (because you still have the vector backbone in the mix).

What made the biggest difference for us in terms of RNA yield was to let the IVT go over night.
After the IVT, NaAc precipitation yields most RNA, but was somewhat toxic in embryo injections in our hands. LiCl precipitation works as well, but we sometimes had problems when washing the pellet. I would highly recommend to isolate the RNA using columns (e.g. Megaclear).It is difficult to obtain very high RNA concentrations after column purification (~ typical concentration is between 70- 150 ng/ul ), but it works for us.

On Thursday, July 11, 2013 12:01:01 PM UTC-4, agnik dasgupta wrote:

[Neal Amin]

Ill throw in my experience here, too.  I used NEB's T7 Quick High Yield RNA synthesis kit.  My template was generated by PCR from the px330 vector (as described in the Jaenisch paper). The 120bp PCR product was PCR purified and P:C:I extracted (not gel purified since I had only one band on PCR), and I used 1ug template in a 30uL reaction as the instructions suggested for small transcripts. 4 hour incubation time.  I performed DNase treatment and used Megaclear columns to purify.  I was skeptical of using these columns since they are LiCl based and LiCl purification is supposedly not effective for small RNAs.  However, when I eluted with 2x50uL of injection buffer, my concentration (by nanodrop) was over 500ng/uL.  This meant a total yield of over 50ug RNA, more than I could ever use.  I also ran the RNA on a gel to check size and to see if the nanodrop concentration matched up with the intensity of the band (excess free nucleotides could have been a reason for the high nanodrop reading), and it indeed did.  Hope this helps.