DNA preparation for microinjection - (Apr/03/2005 )

Hello everyone,

I have some questions regarding the preparation of DNA for Tg mouse generation. I haven't done this before and have some problems regarding the quantity of the DNA. I use 50ug of plasmid DNA for restriction digestion and after gel purification of the fragment, there is only ~300ng left. The fragment is not small (7kb of a 10kb plasmid) and I don't really understand where is it lost. Is that normal? After cutting, I use the Qiagen PCR Purification Kit and then gel purify (Qiagen Gel Extr kit).

Also is that the correct way to submit a DNA fragment for microinjection. Just cut out and purify. I mean shouldn't I blunt-end the overhangs or something?

Thanks really for any suggestions.


Best Regards to All,

Tau

ok, firstly, I just wanna make sure that you used 50 micrograms of DNA. That is a LOT of DNA.

Secondly, What is the capacity of the columns in your Qiagen kits?

Thank you for the reply.

Yes, I used 50ug. The capacity of the Qiagen columns is 10ug, so for the initial digestion mix I used 5 columns. I know it is a lot of DNA, that is why I cannot comprehend why the end-product is so little. The first time I made it, I used 20ug, and the end-product was also ~300ng. So, it just might be the limit of the purification methods (Qiagen's kits). I cannot think of any other explanation.

Any ideas?