1 reply to this topic

[charles_duhwin]

Hey,

I use Ambion's T7 in vitro transcription kits to synthesize cRNA for microinjecting oocytes. I use acidic phenol:chloroform extraction to purify my cRNA. I've always been puzzled by the kit's recommendation to treat the in vitro reaction mix with DNase I to remove the template DNA for two reasons:

1) the DNA template concentration in the reaction mix is very low, so unlikely to interfere with the quantification step too much
2) the DNA partitions to the organic phase during the precipitation step, so should be largely removed anyway

I understand the necessity of the step for down-stream processess like RT-PCR, or if you use a different purification step, but microinjection? The Ambion website recommends the DNase I tep for both RT-PCR and microinjection. Can anyone tell me why this is really necessary?

Thanks,
C

[bob1]

So that it can not be argued that the signal you are seeing is the result of the DNA used to synthesise the cRNA.