Jing Jie Thermal Receipt Printer Driver 253: Troubleshooting and Support Tips

The PCR amplification mixtures of the TCR genefamilies with in-frame and out-of-frame CDR3 lengths were amplifiedin a final volume of 50 µl, containing 4 µl first-stand cDNA, 0.8µl forward AV or BV primer (100 µM), 0.8 µl unlabeled reverse AC orBC primer (100 µM), 5 µl Taq PCR buffer (10X), 4 µl dNTP mixture(2.5 mM), 0.5 µl Taq DNA polymerase (Takara Biotechnology Co.,Ltd.) and 34.9 µl DEPC water. Primer sequences are listed inTable I. The thermal cyclingparameters used were the same as those described for the PCRamplification of TCR AV and BV gene families. The PCR products wereelectrophoresed on a 1.5% agarose gel, stained with 10 µg/mlethidium bromide, and analyzed under ultraviolet light. They werethen purified using a gel extraction kit (Axygen; CorningIncorporated, Corning, NY, USA). The purified PCR products wereligated into the pGEM-T easy vector (Promega Corporation) under theconditions of a 16C water-bath overnight according to themanufacturer's protocol. Ligation products (10 ng/µl; 5 µl) weregently added to 50 µl (5107 cells) competent DH5αEscherichia coli (Takara Biotechnology Co., Ltd.) and werechilled on ice for 30 min, incubated at 42C for 90 sec and chilledon ice for 3 min. SOC medium (800 µl; Takara Biotechnology Co.,Ltd.) was added to the competent cells and cells were cultured at37C in a constant temperature incubator with a speed of 150rpm/min for 50 min. The bacterium solution was centrifuged at 2,500 g for 5 min at room temperature, 900 µl supernatant was discardedand the bacteria were resuspended using the rest of medium, addedevenly to the LB plate (10 g/l tryptone; 5 g/l yeast extract; 10g/l NaCl; 15 g/l agar power; all from Beijing Solarbio Science&Technology Co., Ltd., Beijing, China) containing 100 µg/mlampicillin (Sigma-Aldrich; Merck KGaA) and cultured at 37C for 15h. Positive clones were selected using ampicillin and nucleotidesequences were determined by Genescript Co., Ltd (Nanjing,China).

Jing Jie Thermal Receipt Printer Driver 253

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