Biology Reference
In-Depth Information
12. Aspirate the medium from the target cells and subsequently
add the virus-containing medium. If target cells were seeded
on a 10 cm plate use 10 mL, for 6 wells use at least 2 mL.
13. Incubate the target cells at 37 °C, 10 % CO
2
overnight (S2).
14. (Day 5) Remove the virus-containing medium from the target
cells, wash twice with PBS and supply fresh medium.
15. If a vector expressing a living color reporter gene such as EGFP
was used, expression of the reporter can be monitored 24 h
after infection via fl uorescence microscopy. A EGFP lentiviral
vector provides not only a useful control for transient transfec-
tion effi ciency of packaging HEK 293T cells but also for the
target cell transduction effi ciency.
16. If required, start selecting the cells using the minimum con-
centration of puromycin that causes complete cell death within
5-10 days (
see
Note 22
).
17. MiRNA expression can be monitored, i.e., via qRT-PCR as
early as 6 h post transduction and reaches maximum expression
levels after 24 h.
6
Notes
1. It is advisable to use a large container, as long as it fi ts in the
microwave to reduce the chance of boiling over agarose. Be
aware of boiling delay.
2. Pour the gel slowly because in this case most bubbles stay up
in the fl ask. If any bubbles are formed, push them to the side
using a disposable tip.
3. We recommend storing it at room temperature. It is best fl am-
ing the bottle each time it is opened.
4. Use Stbl3™
E. coli
for transformation, as this strain is particu-
larly well-suited for cloning unstable DNA such as lentiviral
DNA containing direct repeats.
5. MiRNA sequences with more than three Ts or As are not com-
patible with the sh-miR expression strategy, because premature
termination of the transcript will be induced by the Ts.
6. In this mix the fi nal concentration for the oligonucleotides
reaches approx. 107 ng/
L.
7. Annealing occurs most effi ciently when the temperature is
slowly decreased after denaturation because the single oligo-
nucleotides form a hairpin structure.
8. For ligation optimal results, use a 10:1 M ratio of sh-miR
insert:vector and a 3-5:1 M ratio of pri-miR amplicon:vector.
9. Ligation at 16 °C overnight may result in a higher yield of
colonies.
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