dsDNA oligonucleotide cloning for amiRNA expression – Molnar et al. 2008
dsDNA oligonucleotide cloning for artificial miRNA expression
The target sequence is scanned for potential artificial miRNA target sites
(http://wmd2.weigelworld.org/cgi-bin/mirnatools.pl) and long DNA oligonucleotides for
pChlamiRNA2, pChlamiRNA3 and pChlamiRNA3int are designed accordingly
(http://wmd2.weigelworld.org/cgi-bin/mirnatools.pl) and synthesized (Sigma).
A. Oligo Annealing:
(1) Resuspend forward and reverse single-stranded DNA (ss DNA) oligo (90 nucleotide)
in sterile water to the final concentration of 100 µM.
(2) Mix 20 µl of 2X annealing buffer (20mM Tris pH 8.0, 2mM EDTA, 100mM NaCl)
with 10 µl of 100 µM forward and reverse ss DNA oligo.
(3) Boil the mixture in hot water bath for 5 min and cool gradually overnight.
(4) Purify the double-stranded DNA (ds DNA) oligo next day by Qiagen PCR clean up
kit to reduce high salt concentration in annealing buffer. Elute the DNA from the column
with 40 µl of elution buffer and repeat the elution with the flow through.
B. ds DNA oligo phosphorylation:
(5) Set up the phosphorylation reaction as follows
1 µl 90 mer ds DNA oligo (from step 4; ~0.7 µg/µl)
1 µl 10X Promega T4 DNA ligase(!) buffer
1 µl T4 PNK (10 u/µl)
7 µl sterile water
(6) Incubate for 30 min at 37oC, and then inactivate the PNK by placing the tube for 20
min at 65°C.
C. Cloning
(7) Digest the amiRNA vector with SpeI restriction enzyme
10 µl amiRNA vector (~2 µg)
3 µl 10X Buffer H
1 µl SpeI (10 u/µl)
16 µl water
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dsDNA oligonucleotide cloning for amiRNA expression – Molnar et al. 2008
(8) Incubate at 37°C for 1 hr. Heat inactivate the enzyme at 65°C for 20 min then put the
reaction tube on ice.
(9) Dephosphorylate the vector by adding 0.5 µl CAIP (40 u/µl, Cambio). Incubate at
37°C for 30 min.
(10) Gel purify the digested and dephosphorylated vector using Qiagen gel extraction kit.
Elute the DNA with 30 µl of pre-heated buffer. Repeat the elution with the flow through.
(11) Ligate the DNA using Rapid DNA ligation kit (Roche). Set up two ligation reactions
with the diluted phosphorylated ds DNA oligo.
1 µl digested and dephosporylated amiRNA vector (~20 ng/µl)
1 µl 10X or 100X (!) diluted phosphorylated ds DNA oligo (in water)
1 µl 5X DNA dilution buffer
2 µl sterile water
5 µl 2X DNA ligation buffer
0.5 µl T4 DNA ligase
(12) Incubate the ligation reactions at room temperature for 10-15 min.
(13) Transform 100 µl super-competent DH5- cells (home made) with 2.5 µl of ligation
reaction. Select the colonies on LB carb50 plates.
D. Colony PCR to screen amiRNA clones with right orientation
(14) Fill the wells of a PCR tube strips with 80 µl of PCR mixture (10 µl/well)
8 µl 10x PCR buffer (Roche with magnesium)
1.6 µl 10mM dNTPs
0.8 µl 100µM primer AmiRNAprecfor (5’-GGTGTTGGGTCGGTGTTTTTG-3’)
0.8 µl 100µM primer Spacerrev (5’-TAGCGCTGATCACCACCACCC-3’)
3.0 µl 1% W-1
0.8 µl Taq polymerase (5 u/µl)
65 µl dH2O
80 µl
(15) Pick individual colonies from the agar plate using a pipette tip and restreak on a
reference plate. Transfer the same pipette tips to the wells of the PCR tube strips filled
with the PCR mixture and resuspend the remaining colony by pipetting up and down.
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dsDNA oligonucleotide cloning for amiRNA expression – Molnar et al. 2008
Cycling conditions:
95 oC for 2:00
95 oC for 0:30
65 oC for 0:30
72 oC for 0:30
35 cycles
(16) Aliquot 5 µl of 2X loading dye into a fresh PCR tube strip.
(17) Add 5 µl PCR mix to the loading dye and run the resulting 10µl out on a 2% agarose
gel (expected size of a PCR product corresponding the right orientation is 182 bp; wrong
orientation: no product).
E. Miniprep
(18) Extract DNA from 10 ml of overnight culture using Qiagen Miniprep kit.
(19) Elute the DNA with 50 µl of Elution Buffer. Repeat the elution with the flow
through.
F. Sequencing
(20) Set up the sequencing reaction (always use DMSO and high temperature for
annealing because of the strong secondary structure of the miRNA precursor).
2.0 µl DNA from the plasmid prep
1.6 µl (2µM) primer AmiRNAprecfor
2.0 µl BD buffer (5X)
1.0 µl BIGDYE v.3.1
0.5 µl DMSO
2.9 µl dH2O
10 µl
Cycling conditions:
96 oC for 0:45
96 oC for 0:10
60 oC for 4:00
25 cycles
(21) Submit reactions for sequencing.
(22) Align the sequence obtained to the sequence of Forward amiRNA ss DNA oligo.
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