U6/RNAi construction
1.
Choose region of target:
a. Find a successive GGG in the cDNA of the target gene. Choose the sequence at least 100bp
downstream of the ATG.
b. It is wise to check out the following website for available siRNA sequence for the particular gene:
http://katahdin.cshl.org:9331/rnai/repository/scripts/newmain.pl
c. Select 18-19 bases after the ggg to have a 21-22 nt sequence
d. Ideally, the 21-22 nt sequence is 45-65% GC
e. Do the blast to make sure that the selected sequence
U6—G GG…..AAGCTT……..CCCTTTTT G
does not have significant homology to other
-C CC…..TTCGAA....GGGAAAAA CTTAA
Hind III
EcoRI
sequences; and mouse/human conserved; and make
Transcribed
siRNA:
sure it does not have a NotI or EcoRI site.
5’ GGG…… ..A
2. Design the oligo:
3’ CCC……...T
a. the native U6 promoter contains 3 Gs from which
Figure 1 . Two ~50nt oligos needs to
transcription is initiated.
annealed together and inserted i n pBS/U6 .
b. ApaI site is located immediately after the U6
Blunted ApaI will leave 1 G, so only 2G is
promoter.
needed. Use EcoRI as cutt er.
c. TTTT is a PolIII termination signal.
d. Add EcoRI sticky end at the end.
e. Make sure the oligos form hairpin prior to the order.
Examples (Pink is the hairpin sequences: Blue is the complementary hairpin seq; black is the common sequence
for all oligos).
OLIGO F: ggattcatgaatggcccttaAAGCTTtaagggccattcatgaatccC TTTTT G
OLIGO R: aattc AAAAA GggattcatgaatggcccttaAAGCTTtaagggccattcatgaatcc
OLIGO F: ggtctgctgagtccgcagcaAAGCTTtgctgcggactcagcagaccCTTTTT G
OLIGO R: aattc AAAAAGggtctgctgagtccgcagcaAAGCTTtgctgcggactcagcagacc
3.
Anneal and ligate with pre-processed pBS/U6 vector as shown in the figure (note: digestion with ApaI should
be at RT):
a. Dilute the primers at 100µM and then in 20 µl annealing mix, add 2.5µl of each primer and add 15µl
H2O to 20µl.
b. Using a PCR program, start from 95C and
ApaI-XhoI-SalI-HindIII -RI
then reduce 5C every 5min to 30C.
U6
c. Measure concentration about 600ng/ml
promoter
and add 180µl H2O to make it 60ng/ml.
Two oligos
annealed
d. Use 1µl of vector (ApaI (4/BSA, RT)HindIII
pSuperklenow blunt-gel purify- EcoRI-gel purify), SalI
U6p-puro
XhoI
0.5µl insert in a 10µl ligation reaction.
Ligate 1hr or overnight if using the
incompetent cells.
ApaI
e. Use 5µl for transformation (Ampr). There
Klenow
are not necessarily more colonies on the
EocRI
ligation than controls- just pick 4-6, and you
should get 1-2 positive ones.
f. If using pSuper-Retro-Puro-U6 promoter as
vector for retroviral construct, screen by
Ligate
XhoI (linear) and HindIII (300bp). In both
Transform
(Amp r)
cases, both digestions should be right.
g. There is no need to sequence because
sequencing will stop right before the hairpin.