Zero blunt topo pcr cloning vector

In total, 1 μl of each cell pellet was subjected to genomic DNA extraction by treating with 20 μL of QuickExtract DNA Extraction Solution (# QE09050, Epicenter, USA) at 65 °C for 6 min followed by heat inactivation at 98 °C for 2 min. In all, 1 μl each of these treated samples was used for genomic PCR to amplify a flanking region of the 3rd exon of DDX4, where the gRNA sequences were designed, using high fidelity HIFI PCR premix (Clonetech, USA) with primers summarized in the resource table. The resultant PCR products were either separated by agarose gel electrophoresis and visualized or subjected to sub-cloning into the Zero Blunt TOPO PCR Cloning Vector (Invitrogen, USA) for sequencing.

Rapid Amplification of cDNA Ends (5′-RACE) PCR was performed on 5 µg of total RNA isolated from H2126 cells using the Generacer™ kit (Invitrogen). Briefly, cDNA prepared using the Generacer™ kit (Invitrogen) was amplified using a B₁R gene specific reverse primer RT Rev 3, and GR 5′Primer, a forward primer that anneals to ligated Generacer™RNA oligo. Nested PCR was performed using GR5′nested and RTRev2 primers (conditions as above). PCR products were subsequently analysed using gel electrophoresis before cloning into Zero Blunt TOPO PCR cloning vector (Invitrogen) using a Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen) according to the manufacturer's protocol. The TOPO cloning reaction (4 µl PCR product, 1 µl salt solution and 1 µl of TOPO vector) was mixed and incubated for 5 min at RT and used for transformation of TOP10 E. coli chemically competent cells (Invitrogen) using heat-shock transformation. Transformed cells were plated on Luria Bertani (LB) agar plates containing ampicillin (50 µg/mL).
Single colonies were inoculated and grown overnight in LB media containing 50 µg/mL ampicillin at 37°C and plasmid DNA was extracted using Plasmid-Mini kit (Qiagen) following manufacturer's instructions. Plasmid DNA containing 5′ RACE nested PCR products were sequenced, using B₁R primers (Table 2) to identify the 5′end/s of B₁R.

Mapping was performed according to the 3′-RNA ligase-mediated RACE method^{31 (link)} with minor modifications: Total RNA preparations were first 3′-dephosphorylated using T4 PNK for 1 h at 37 °C without ATP and pre-adenylated linker (Universal miRNA cloning linker, NEB) ligation was performed during 4 h at 22 °C in the presence of truncated RNA ligase 2 (NEB)^{30 (link)}. Reverse transcriptase reactions were performed using primer prE complementary to the linker sequence. PCR primer prF specific to mRNA1, or primer prK specific to (CGA)₄-mRNA, were used with primer prE in PCR reactions (Supplementary Figs. 3a and 6c). PCR products were purified, cloned into Zero Blunt TOPO PCR Cloning vector (Invitrogen), transformed and plasmids sequenced.