Generuler 100 bp plus dna ladder

The specificity of the PCR reaction was assessed with agarose gel electrophoresis and melting curve analysis. For each candidate reference gene PCR products using HCT 116 samples were analyzed with agarose gel electrophoresis with 2% agarose and TAE running buffer. For each gene 2–5 µl of PCR products were mixed with loading dye and loaded on the gel. GeneRuler 1 kb Plus DNA Ladder and GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific SM0321) were used as markers. Gel Doc XR + Imager was used for imaging. Melting curve analysis was routinely performed after amplification. Technical replicates of qPCR reactions were excluded in case of aspecific product formation according to the melting curve analysis.

For balanced translocations between TRAC and B2M/CIITA, we initially observed a background signal for ddPCR assays with a forward primer binding to the TRAC homology arm of the HDRT, leading to false-positive events in ddPCR 4 days after transfection (TRAC_B2M, Additional file 1: Fig. S6a,b,c). When the T cells were expanded until day 14, the noise disappeared, presumably due to degradation of the HDRT (Additional file 1: Fig. S6 a,e,f). When the reporter probe was placed on the B2M instead of the TRAC locus, the background was eliminated (Additional file 1: Fig. S6 b,g,h). Overall, at day 4 after transfection, no significant difference was observed in the total translocation frequency between unedited (0.05%, SD = 0.06) and TRAC-CAR KI (Cas12a) + MHC dKO (nCas9-BE) samples (Fig. 5f). The specificity of the assays were validated by PCR of genomic DNA from TRAC-KO (Cas9) + MHC dKO (Cas9) (triple KO) samples and synthetic gene fragments (gBlocks™ (gB) by Integrated DNA Technologies Inc) modeling the translocations as positive controls (Additional file 1: Fig. S6i) The test PCRs were performed using Red Taq DNA Polymerase Master Mix (VWR) and the GeneRuler™ 100 bp Plus DNA Ladder (Thermo Fisher Scientific).

Clinical samples spotted on FTA cards, in which parasites were identified by cyt b gene analysis in a previous study, were subjected to PCR-RFLP analysis. PCR amplifications targeting mpi and 6pgd were performed as described above using a high fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan). The PCR products were digested by restriction enzymes HaeIII, HapI, and BstXI for the mpi gene and Bsp1286I and HinfI for the 6pgd gene, and resulting restriction fragment patterns were analyzed by 2% agarose gel electrophoresis. GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific, Waltham, MA) was used as a DNA size marker. The gel was stained with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA), and DNA fragments were visualized with UV transilluminator.
Differentiation between L. (V.) guyanensis and L. (V.) panamensis was performed by restriction enzyme-digestion of the hsp70 gene fragment [27 (link)]. Briefly, the hsp70 gene fragment was amplified by a nested PCR using sets of outer primers (L.HSP-Ty1S and L.HSP-OR) and inner primers (L.HSP-Ty2S and L.HSP-IR2) (Table 2). The amplicons were digested with a restriction enzyme, BccI, and resulting fragment patterns were analyzed by 3% agarose gel electrophoresis.

The specificity of the PCR products was tested first using gel electrophoresis with 2% agarose gel in TBE buffer stained with GelRed nucleic acid gel stain (Biotium). GeneRuler 100 bp plus DNA ladder (Thermo Scientific) was loaded as marker. Gel Doc XR+ Imager (Bio‐Rad) was used for imaging. To confirm the identity of all PCR products—except for the PCR product for the nuclear and mitochondrial isoforms, which were too short for sequencing—DNA was isolated from agarose gel with NucleoSpin gel and PCR clean‐up (Macherey‐Nagel, Duren, Germany) according to the manufacturer’s recommendations and sequenced by Microsynth Seqlab, Göttingen, Germany. To confirm the identity of the PCR products for the nuclear and mitochondrial isoforms, PCR product for both isoforms with Rev4 reverse primer using ovary cDNA was isolated from agarose gel. The concentration of the DNA was determined with NanoDrop ND‐2000, and the samples were used as template in a second round of PCR with 10 and 0.1 fg·µL⁻¹ final concentration for both isoforms. For further reactions, the specificity of the formed PCR products was confirmed with melting curve analysis. Results were accepted only with uncompromised product specificity—or indicated otherwise for the process of optimization.

Bacterial genomic DNA samples were extracted using ZymoBIOMICS DNA Kits (Zymo Research, CA, USA), following the manufacturer’s instruction. The PCR reaction mixture (25 µL) contained a 1× JumpStart REDTaq ReadyMix (Sigma) and each primer (Table 2). The list of primers used in the mPCR are also presented in Table 2 [34 (link),35 (link),36 (link),37 (link)]. The PCR reaction was performed as follows: initial activation of DNA polymerase at 95 °C for 3 min, plus 35 cycles of denaturation at 95 °C for 30 sec, primer annealing and extension at 62.5 °C for 1.30 min, and a final extension at 72 °C for 5 min. A negative control was included in each run, consisting of the same reaction mixture but with water instead of template DNA.
The PCR products (5 µL) were analyzed using gel electrophoresis on 1.5% (w/v) agarose gel in 0.5 × TBE buffer at a constant voltage of 100 V for 30 min (Mupid exU system; Takara; Tokyo, Japan). The gels were stained with ethidium bromide and visualized under ultraviolet light (GeneGenius Bioimaging System; SynGene; Cambridge, UK). The sizes of the PCR products were determined by comparison with molecular-sized standards (GeneRuler™ 100 bp Plus DNA ladder; Thermo Fisher Scientific; Vilnius, Lithuania).