Genomic DNA from ∼106 parental or sorted CEM/R5 cells or CD4 lymphocytes was purified using a Quick-gDNA MiniPrep kit (Zymo Research, USA). The CXCR4 target regions were PCR amplified using 200 ng of genomic DNA, Taq DNA polymerase (SibEnzyme, Russia), and primers listed at the end of Table S2 . PCR was set up using the following parameters: (95°C, 2 min) once, (95°C, 30 s; 65°C, 30 s; 72°C, 30 s) 30 times, and (72°C, 5 min) once. The DNA fragments amplified from double-positive cells and containing an integrated transgene were resolved on agarose gel, purified, and cloned into pJet 1.2 PCR cloning vector (Thermo Scientific, USA). The DNAs from single bacterial clones were sequenced using the Sanger method. The level of indel formation in the PCR-amplified target locus was analyzed by T7 endonuclease assay using a standard protocol.
Pjet 1.2 pcr cloning vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PJet 1.2 PCR cloning vector is a commercially available plasmid designed for the cloning of PCR products. It provides a simple and efficient method for the direct insertion of PCR amplicons into the vector. The vector contains a multiple cloning site for the insertion of the PCR product and confers ampicillin resistance for selection purposes.
Automatically generated - may contain errors
3 protocols using pjet 1.2 pcr cloning vector
1
Genomic DNA Extraction and CXCR4 Amplification
2
Genomic DNA Extraction and Mutation Analysis
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Genomic DNA from 3 × 106 parental or sorted 293 T cells was purified using Quick-gDNA MiniPrep Kit (Zymo Research, USA). The VDAC1 and VDAC3 target regions were PCR-amplified using 200 ng of genomic DNA, Pfu DNA polymerase (SibEnzyme, Russia) and primers listed in Supplementary Table 2 . The DNA fragments amplified from double positive cells and containing integrated transgene were resolved on agarose gel, purified and cloned into pJet 1.2 PCR cloning vector (Thermo Scientific, USA). The DNAs from single bacterial clones were used for Sanger sequencing. The DNA fragments corresponding to target locus with no integration were purified as outlined above. Indels formation in these fragments was determined using Surveyor Mutation Kit (Transgenomic, USA) and in accordance to manufacturer’s protocols.
3
Semiquantitative Fluorescent RT-PCR Analysis
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Retrotranscription was carried out with 400 ng of RNA and the RevertAid First Strand cDNA Synthesis Kit (Life Technologies), using the vector-specific primer RTPSPL3-RV (5'-TGAGGAGTGAATTGGTCGAA-3'). Samples were incubated at 42°C for 1 hour, followed by 5 min at 70ºC. Then, 40ng of cDNA (final volume of 50 µl) were amplified with SD6-PSPL3_RT-FW (5'-TCACCTGGACAACCTCAAAG-3') and RTpSAD-RV (Patent P201231427) (size 1,016 nt) using Platinum-Taq DNA polymerase (Life Technologies). Samples were denatured at 94ºC for 2 min, followed by 35 cycles of 94ºC/30 sec, 60ºC/30 sec, and 72ºC (1 min/kb), and a final extension step at 72ºC for 5 min. RT-PCR products were sequenced as previously indicated. Rare transcripts were subcloned into the pJet1.2 PCR cloning vector (Thermo Fisher Scientific) and sequenced.
In order to relatively quantify all transcripts, semiquantitative fluorescent RT-PCRs were undertaken in triplicate with primers PSPL3_RT-FW and RTpSAD-RV (FAM-labelled) and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied [20] . FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0. Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.
In order to relatively quantify all transcripts, semiquantitative fluorescent RT-PCRs were undertaken in triplicate with primers PSPL3_RT-FW and RTpSAD-RV (FAM-labelled) and Platinum Taq DNA polymerase (Life Technologies) under standard conditions except that 26 cycles were herein applied [20] . FAM-labeled products were run with LIZ-1200 Size Standard at the Macrogen facility and analyzed with the Peak Scanner software V1.0. Only peak heights ≥ 50 RFU (Relative Fluorescence Units) were considered.
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