Biology Reference
In-Depth Information
5. For the cloning of psiCHECK-miR122, a control ligation
reaction without insert should also be performed and trans-
formed into NovaBlue competent cells. If there are back-
ground colonies on the control plate, an additional digest with
Xho
I can be performed using the ligation products to remove
any re-circularized psiCHECK-2 plasmid without insert.
6. The Huh7-psiCHECK-miR122 cell line should be cultured in
DMEM supplemented with 10 % FBS, 2 % penicillin/strepto-
mycin, and the concentration of G418 determined by the via-
bility experiment (
see
Subheading
3.4
).
7. The 500
g/mL of G418 stated here can act as a guideline for
Huh7 cells; however, the viability experiment should be per-
formed using the cell line of interest (including Huh7 cells)
and the specifi c G418 batch to be used for selection.
8. Because the stable cell line is developed by co-transfection of
psiCHECK-miR122 and the pcDNA3 plasmid, some clones
will incorporate only the pcDNA plasmid and will therefore
show G418 resistance but no luciferase expression.
μ
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