Pcr thermal cycler

Polymerase chain reactions (PCR) were performed in a PCR thermal cycler (Bio-Rad, USA) with 50 μl of reaction mixture containing the template (1 μl), each primer (2 μl), polymerase (1 μl), dNTP (1 μl), deionized water (18 μl), and 10× buffer (25 μl). The PCR was run following this program: pre-denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 15 sec; annealing at 57°C for 1 min and extension at 72°C for 1min.
The flag-capA gene was cloned by PCR using specific primers capA-F1/capA-R1 (capA-F1:5'-AAGGACGAC GATGACAAGGATCAAGCAGTAAGTTTTGACTTTTTTATCCGG-3' and capA-R1:5'-TTCAGACTTTGC AAGCTTCTAAACTCGTTTCATCGCTTGTGACG-3') (Table 1). The PCR fragment containing flag-capA gene was cloned into vector pMG36e to generate new plasmid pMG36e-flag-capA. The new resultant plasmid was transformed into L. plantarum P1 by electroporation at 1.25 kV/cm in a Bio-Rad GenePulser, and then the cells were diluted with 400 µl of regeneration medium MRS. The competent cells of L. plantarum P1 were prepared following the protocol below. L. plantarum P1 cells were cultured in MRS at 30°C overnight. A total of 50 mL MRS with 1% glycine and 0.75 M sorbitol was inoculated with 1% of the overnight culture. At OD₆₀₀ of 0.3, the cells were harvested by centrifugation at 6,000 ×g, and then washed by sterile water for three times and stored in 1/125 30%PEG3000 [33 (link)].

A millilitre (1 ml) of the venous blood samples of the 300 acute lymphoblastic leukemia (BCP ALL and T cell ALL) patients 15 days after remission induction were collected in the K₃EDTA-coated tubes. The DNA extraction from blood samples patients was done according to the instructions of Sam brook 2001 organic protocol¹³ and stored at − 20 °C until used. The SNPs were screened from dbSNP^{14 (link)} and their presence in the Pakistani population was confirmed by the Ensemble genome browser^{15 (link)}. To examine the polymorphism, the Tetra-ARMS primers were designed by using PRIMER 1 online software (http://primer1.soton.ac.uk/primer1.html)^{16 (link)} (Supplementary Table S1) and amplification was performed by using PCR thermal cycler (Bio-Rad^®, USA) following gel electrophoresis.

RNA was isolated from cells or brain tissues with an RNA isolation kit (Vazyme, China) according to the manufacturer's specifications. Complementary DNA (cDNA) was synthesized using a PCR Thermal Cycler (Biorad, USA) with the HiScript cDNA kit (Vazyme, China). The qRT-PCR gene expression analysis was performed using ChamQ Universal SYBR qPCR Master Mix Kit (Vazyme, China) on Step One Plus (Applied Biosystems, Waltham, MA) with the appropriate primer pairs (Table S2). qRT-PCR data were normalized to GAPDH as housekeeping standard. Fold changes of target mRNAs were analyzed using the 2 ^−ΔΔCT method.

The primer sequences used in this study are listed in Supplemental Table 1. Total RNA extraction, reverse transcription and qRT-PCR were conducted as previously described [10 (link)]. The tissues were cut into 0.5 cm³ pieces and sequentially triturated in liquid nitrogen, while the cells were digested in 0.1% trypsin (Gibco, Life Technologies, Carlsbad, CA, USA). Then, total RNA extraction was performed using TRIzol (Invitrogen) according to the manufacturer's protocol. After confirming the amount and purity of total RNA using Biophotometer plus (Eppendorf, Germany), the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, USA) was used to reverse-transcribe total RNA to cDNA. cDNA was amplified using PCR Thermal Cycler (Bio-Rad, USA) by first heating at 65℃ for 10 min, incubating at 55℃ for 30 min, deactivating at 85℃ for 5 min and finally storage at 4℃ for 5 min. qRT-PCR was performed by SYBR Master Mix (Roche Applied Science) using a reverse transcription system (LC-480, Roche, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression data.

The total RNA obtained from the purified peripheral γδT cells was converted to complementary DNA (cDNA) using an Evo M‐MLV RT kit with gDNA cleaned for qPCR II (AG111728, Accurate Biotechnology). PCR experiments were performed in a PCR thermal cycler (Bio‐Rad). GAPDH served as the internal control. Primer sequences are listed in Supporting Information: Table 2.