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
biotech, on Sep 17 2004, 11:52 AM, said:
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!