Following heat treatment and centri

Following heat treatment and centrifugation, the supernatant was used for Pfu activity analysis. The relative DNA polymerase activities were determined by comparing the band intensities of a PCR-amplified DNA fragment obtained using the His6-tagged Pfu DNA polymerase with the commercial Pfu DNA polymerase (Cwbio, China) [7, 8]. The primers [sense [5’- TCCTGCTCGCTTCGCTACTT-3’] and antisense [5’- CGCATTCACAGTTCTCCGCA-3’]] amplified a 1 kb fragment from the pACYC184 plasmid. The PCR reactions consisted of a 25 μL mixture containing 1 μL of plasmid DNA (10 pg), 2.5 μL 10x reaction buffer [100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris–HCl (pH 8.8), 20 mM MgSO4, and 1 % Triton X-100], 2 μL dNTP mixture (2.5 mM of each dNTP), 1 μL each primer (10 lM), 1 μL non-purified Pfu DNA polymerase or 1 μL commercial Pfu DNA polymerase (final concentration of 0.6 U μL-1) diluted with sterilized ultrapure water, and 16.5 μL sterilized ultrapure water. The PCR was performed using an automated thermocycler (T100 Thermocycler, Bio-Rad) at 95oC for 2 min; 30 cycles of 95oC for 30 s, 60oC for 30 s, 72oC for 1 min; 72oC for 5 min. 5 lL PCR mixture was separated using electrophoresis on a 1 % agarose gel in 1x TBE buffer. Following ethidium bromide staining, the DNA bands were visualized, and the band brightness was analyzed (ChemiDoc XRS+ with Image Lab Software, Bio-Rad). The nonpurified Pfu DNA polymerase were diluted with deionized water until the relative DNA polymerase activities were within the calibration curve range of 0.4–0.9 U (y = 5.731x 10-8x + 0.3025, R2 = 0.992, y is the activity of Pfu DNA Polymerase, x is the image volume of band brightness).
Samples were taken at different time points after adding the IPTG, and the cells were collected by centrifugation at 14,000g, 4oC for 10 min. The supernatant was filtered using a 0.22 lm filter, diluted with deionized water until the IPTG concentration in the medium was within the linear range of 5–1,000 μM, and then stored at -20oC until analysis. The cell pellet was washed three times with deionized water and adjusted to an OD600 value from 3 to 4 with deionized water. The cell suspensions were flash frozen using liquid nitrogen and stored at -80 C until analysis. For analysis, the cell suspensions were thawed on ice and sonicated (Digital Sonifier 450, Branson Ultrasonics) with 15 s pulses followed by 2 min intervals on ice four times. The crude extract was collected by centrifugation at 14,000g, 4oC for 10 min. The supernatant was filtered using a 0.22 lm filter before IPTG analysis. The IPTG analysis method was modified from the reported method [9] and performed on an Agilent HPLC with an UV/Vis detector operating at 210 nm. Samples were kept in the auto-injector. The mobile phase was 5 mM H2SO4, and the flow rate was 0.6 mL min-1. Ten microliters of the samples or standards were directly injected into a 300 mm 9 7.8 mm HRX-87H Ion Exclusion column (BioRad, USA). The samples, column, and auto-injector were kept at 25 C. The retention time of the IPTG was 11.5 min.

Kết quả (Việt) 1:

Sau xử lý nhiệt và số, supernatant đã được sử dụng cho Pfu hoạt động phân tích. Các hoạt động tương đối DNA polymerase đã được xác định bằng cách so sánh các cường độ ban nhạc của một mảnh DNA khuếch đại PCR thu được bằng cách sử dụng gắn thẻ His6 Pfu DNA polymerase với thương mại Pfu DNA polymerase (Cwbio, Trung Quốc) [7, 8]. Các chất nền, mồi [cảm giác [5'-TCCTGCTCGCTTCGCTACTT-3'] và antisense [5'-CGCATTCACAGTTCTCCGCA-3']] khuếch đại một đoạn 1 kb từ pACYC184 plasmid. Phản ứng của Đảng Cộng sản Romania bao gồm một hỗn hợp 25 μL có 1 μL plasmid DNA (10 pg), 2.5 μL 10 x phản ứng đệm [100 mM KCl, 100 mM (NH4) 2SO4, 200 mM Tris-HCl (pH 8.8), 20 mM MgSO4, và 1% Triton X-100], 2 μL dNTP hỗn hợp (cách 2.5 mM của dNTP mỗi), 1 μL mồi (10 lM), 1 μL phòng không tinh khiết Pfu DNA polymerase hoặc 1 μL thương mại Pfu DNA polymerase (nồng độ cuối cùng của U cách 0.6 μL-1) pha loãng với bột ultrapure nước và 16,5 μL khử trùng nước ultrapure. Đảng Cộng sản Romania được thực hiện bằng cách sử dụng một thermocycler tự động (T100 Thermocycler, Bio-Rad) tại 95oC cho 2 phút; 30 chu kỳ của 95oC cho 30 s, 60oC cho 30 s, 72oC cho 1 phút; 72oC cho 5 phút 5 lL PCR hỗn hợp đã được tách ra bằng cách sử dụng điện trên một agarose 1% gel trong 1 x TBE bộ đệm. Sau ethidium bromua nhuộm, các ban nhạc DNA đã hình dung, và độ sáng ban nhạc được phân tích (ChemiDoc XRS + với hình ảnh phòng thí nghiệm phần mềm, Bio-Rad). Nonpurified Pfu DNA polymerase được pha loãng với nước deionized cho đến khi các hoạt động tương đối DNA polymerase trong phạm vi đường cong hiệu chuẩn của 0.4-0,9 U (y = 5.731 x 10-8 x + 0.3025, R2 = 0.992, y là các hoạt động của Pfu DNA Polymerase, x là phần hình ảnh của ban nhạc độ sáng).Samples were taken at different time points after adding the IPTG, and the cells were collected by centrifugation at 14,000g, 4oC for 10 min. The supernatant was filtered using a 0.22 lm filter, diluted with deionized water until the IPTG concentration in the medium was within the linear range of 5–1,000 μM, and then stored at -20oC until analysis. The cell pellet was washed three times with deionized water and adjusted to an OD600 value from 3 to 4 with deionized water. The cell suspensions were flash frozen using liquid nitrogen and stored at -80 C until analysis. For analysis, the cell suspensions were thawed on ice and sonicated (Digital Sonifier 450, Branson Ultrasonics) with 15 s pulses followed by 2 min intervals on ice four times. The crude extract was collected by centrifugation at 14,000g, 4oC for 10 min. The supernatant was filtered using a 0.22 lm filter before IPTG analysis. The IPTG analysis method was modified from the reported method [9] and performed on an Agilent HPLC with an UV/Vis detector operating at 210 nm. Samples were kept in the auto-injector. The mobile phase was 5 mM H2SO4, and the flow rate was 0.6 mL min-1. Ten microliters of the samples or standards were directly injected into a 300 mm 9 7.8 mm HRX-87H Ion Exclusion column (BioRad, USA). The samples, column, and auto-injector were kept at 25 C. The retention time of the IPTG was 11.5 min.