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I need to get large amounts of my vector but cannot transform it in E. Coli (due to some special bases), so i needed to optimize my PCR, Digestion and ligation. Now I am currently trying to optimize my ligation.
My Insert is about 500bp big, my vector 5590bp.
I use a mol-ratio of insert:vector 3:1
So i now want to ligate:
1µg Insert
4µg Plasmid
To get the total DNA conentration of about 10µg/mL I want to use a total volume of 500µL and 3µL of T4 DNA Ligase (NEB)
Now my questions
Do you have any experiences ligating large amounts? Do you think 500µL is a volume big enough? How much T4 Ligase would you use?
thans a lot in advance
