Pcr script amp cloning kit

The region between the markers D17Mit21 and 22 contains the following genes: H2-Ab1, H2-Aa, H2-Eb1 and H2-Eb2. To clone the H2-Ab1 and H2-Aa genes, RNA was extracted from spleens using SV Total RNA Isolation System (Promega, Madison, WI, USA) and treated with DNase I (AMPD1,Sigma-Aldrich, St.Louis, USA). cDNA was synthesized with oligo-dT18 primers (Thermo Fisher Scientific, Inc) and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Primer sequences for cloning were selected from the Ensembl database (version GRCm39) for the H2-Ab1, H2-Aa genes of C57BL/6 mouse strain. 5’ forward primers ended up at the ATG start codon; reverse primers started at the TGA stop codon. Coding DNA was amplified using Advantage GC Genomic LA Polymerase (Clontech, Takara Bio, USA, Inc). PCR products were extracted from gels with Cleanup Mini Set (Evrogen, Moscow, Russia) and cloned in the PCR-Script Amp Cloning vector using the PCR-Script™ Amp Cloning Kit (Agilent (Stratagene, Santa Clara, CA,USA) or pAL-TA (Evrogen, Moscow, Russia) after 3 preliminary cycles of PCR products amplification with Taq-polymerase (Helicon, Moscow, Russia). Four-five positive clones were sequenced for each gene.

PCR on cDNA generated from total RNA of knee SF was used to produce an amplicon of 267 base pairs of HOTAIR (see Supplementary Table S4). Amplicons were cloned into pPCR-Script Amp SK (+) plasmids using the PCR-Script Amp cloning kit (Agilent Technologies). Plasmids were amplified and purified with the PureLink MiniPrep kit (Thermo Fisher Scientific). Plasmids were linearized using restriction enzymes (EcoRI or NotI; both New England Biolabs) and purified with the QIAquick PCR Purification Kit (Qiagen). DIG-labeled HOTAIR probes were prepared by in vitro transcription with RNA polymerases and plasmid vectors containing target transcript sequences. Linearized plasmid DNA (10 µl) was used as a template, and RNA probes were synthesized with T7 or T3 RNA polymerase (Roche) and DIG Labeling Mix (Roche) for 100 min at 37 °C.