Biology Reference
In-Depth Information
10. Phenol is a hazardous organic solvent. Always use suitable
laboratory gloves when handling phenol containing solutions.
Specifi c waste procedures may be required for the disposal of
phenol-containing solutions.
11. Carefully pour out or aspirate supernatant (watch the DNA-
pellet, do not lose it!).
12. Failure to immediately add medium to the electroporated cells
can signifi cantly reduce cell viability and decrease transforma-
tion effi ciency.
13. It is useful to plate at least two different aliquots of bacterial
suspension (i.e., 50 and 150
L), in order to increase the
chance to obtain enough single colonies the next day.
14. Don't forget to prepare a glycerol stock for maintaining the
bacteria containing the desired plasmid.
15. The fl anking sequence of a pri-miRNA clone may differ from
the NCBI reference with respect to biological polymorphisms.
This should not affect the function of the mature miRNA.
16. It is very important to have good single cell suspensions (tryp-
sinize well) and to evenly distribute the cells.
17. Prior to use for lentiviral vector production, cells should have
undergone at least 2 passages following thawing.
18. If the chosen vector does not contain a reporter gene such as
EGFP for an easy qualitative estimate of the transfection and
transduction effi ciency of your viral preparations we highly
recommend running a parallel experiment with a lentiviral
vector of your choice expressing such a reporter. This will
enable you to control all major steps of the protocol. It cannot
control for transfection effi ciency of your plasmid containing
the transgene.
19. HEK 293T cells detach easily, be careful, as well with all
medium changes.
20. From this step on, cells are considered as Biosafety Level 2.
21. We recommend not storing viral stock at this step, i.e., at
−80 °C, because viral titers will signifi cantly go down after one
freeze thaw cycle. Furthermore, consider one of the many len-
tiviral titration methods in order to be able to compare virus
production effi ciency from experiment to experiment [
8
].
22. Prior to beginning experiments, determine the concentration
of puromycin for target cells by performing a puromycin kill
curve. Excess of puromycin can cause many undesired pheno-
typic responses in most cell types.
23. The agarose gel is non-denaturing; therefore, the single-
stranded oligos do not resolve at the expected size due to for-
mation of secondary structure.
μ
