you could run the gel to see whether mRNA is presented because the dNTPs also will give you the numbers.

Hi:

I always use the phenol: chloroform purification, and works well.

however, a tech in our lab, he uses the LiCl, also works well.

So, both works.

you could run the regular agarose gel but load the RNA sample with the loading provided in the kit and heat 65oC for 3-5 mins, and run 5-10 min gel to see the RNA. longer time running gel, the RNA will gone.

Hi,

I use the mMessage Machine kit (which is what i think you used as well). There are several factors that can play role here.

1. Run a gel to make sure your plasmid is linearized, because sometimes, that could be problem.

2. Make sure your DNA template concentration that you are adding to your mix is as mentioned in the protocol with the kit. Because I have seen that if there is excess of DNA, it can interfere with the process of IVT.

3. I have always used Lithium Chloride for precipitation and I do add 60ul into my reaction. I did use the same method in my publication as well (LiCl ppt)

(Article Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho Durin...

and I leave it overnight at -80 degree celsius. I do centrifuge at 14000rpm for 45 minutes.

4. Sometimes, the pellet is so small that you can not see it and it can form a smear on the side of the tube. So irrespective of if I see my pellet or not, I take out the supernatant and air dry my tube for about 15-20 minutes and then resuspend it and measur my concentration.

Hopefully, this helps.

Deba