Biology Reference
In-Depth Information
8. Trypsin-EDTA: 0,05 %: commercial source.
9. HEK 293T cells (ATCC, Manassas, VA, USA).
10. Sterile fi lters (0.45
μ
m).
11. 20 mL syringe.
3
Methods
Due to the differences in the cloning procedures required for
generation of pri-miR or sh-miR-expression vectors, experimental
details are given separately for both strategies whenever necessary.
3.1 Design of
Primers for
pri-miRNAs
1. Pre-miRNA and fl anking sequences can be obtained from
http://useast.ensembl.org/index.html
.
2. Design primers complementary to the ends of the pri-miR
region (pre-microRNA (60-70 nucleotides) and their fl anking
200-300 nucleotides) with the following features: Primer
length between 18 and 24 nucleotides. The primers should be
long enough to give a melting temperature (
T
m
) of 55-60 °C.
T
m
s for both primers should be within 2 °C. The GC-content
should be 40-60 %. The 8 bases at the 3
end should preferably
have 5 or more As and Ts to prevent it from being sticky. The
3
′
end itself should be a G or C to improve the effi ciency of
priming. The second and third bases from the 3
′
′
end should be
As and/or Ts.
3. Primer pairs should be checked for complementarity at the
3
-end to reduce primer-dimer formation (no more than 3 to
max. 4 nt complementary to any primer sequence).
4. Extend the 5
′
′
-end (forward) primer with the
Age
I and the
-end (reverse) primer with the
Eco
RI recognition site.
5. To increase cleavage effi ciency, add 4 bases to the 5
3
′
-end of
each restriction site. This is necessary because restriction
enzymes cleave DNA much less effi cient towards the end of a
fragment. An example is shown below:
-Forward primer with
Age
I: 5
′
′
-CGCTACCGGTNNNN
NNNNNNNNNNNNNNNN-3
′
.
-Reverse primer with
Eco
RI: 5
-TCGAGAATTCNNNNNN
NNNNNNNNNNNNNN-3
′
′
.
3.2 Design of
Primers for sh-RNAs
1. Mature miRNA sequence can be obtained from
http://www.
2. Design and synthesize two complementary single-stranded
oligonucleotides as illustrated in Fig.
1
(
see
Note 5
): The sense
strand oligo contains an
Age
I overhang (CCGG) at the 5
end
followed by the anti-mature-, loop-, and mature sequence and
′
