Posted 17 September 2004 - 05:59 AM
The complete protocol is below:
8 microliter vector
10 microliter insert DNA
2 microliter 10X T4 ligation buffer
0.4 microliter T4 ligase.
Incubate at 22 degree for 1 hour;
Inactive T4 by heating the reaction at 65 degree for 10 min.
Bs: I load 10 microliter of the mixture while I run the gel?
Can anybody tell me what I can do ? Thank you very much!!
Posted 17 September 2004 - 10:52 AM
Posted 17 September 2004 - 05:28 PM
Posted 17 September 2004 - 07:36 PM
Firstly thank you very much for your reply!
Well, if you don't see anything on your gel, you either have insufficient amount of DNA to see, or forgot to add ethidium bromide to the gel. You write the volumes of your vector and insert but what is their concentration? Usually, you cannot see more than <100 ng of DNA on an agarose gel, maybe that's the reason?
I use fluorometer to detect the concentration of purified plasmid. It is very low only 15-20ng/ul. The digested insert isn't purified after the double digestion since I loss almost all the DNA while purification. Do you think it is necessary to purify the insert firstly before ligation?
Can I use the ligated mixture for transformation even though I can't see the bands in the gel?
Hope to see your reply soon!
Thanks a lot!