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Cloning into lentiviral vector - (Jul/08/2011 )

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i've normally use STBL3 cells, but I'm not sure what the difference is, sorry.



I am trying to do something similar (ligation 12.4 kb vector and 6.7 kb insert), I am using DH5alpha and in the previous plates I got 4x colonies more in the ligation plates compared to the AP vector but they lack a piece...

This time I did ligation at 1:1 in 10 microliters volume with 50 ngr DNA total, 100 nanogram dna total and 1:2 ratio with 200 ng total... I have seen in other forums that 1:1 was the best ratio and different opinion in the total amount of DNA to be used...

Anyway, grow the cells at 37C for 12 hours then I saw that they should be better grown at 30C so I throw them at that temperature and this morning I got lots of colonies in the AP vector one and fewer colonies in the ligation one.. is this a good sign???

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