Deoxynucleotide triphosphates dntps

All primers and plasmids were synthesized by Sangon Biotech (Shanghai, China); (NH4)₂SO₄, KCl, KOH, Tween 20, and neutral red were purchased from Sigma-Aldrich (Shanghai, China). Deoxynucleotide triphosphates (dNTPs) and MgSO₄ were obtained from New England Biolabs Ltd. (Beijing, China). Glycerol-Free Bst polymerase (MDX018-08A) was purchased from Meridian Biotech Ltd. (Shenzhen, China); the DNA extraction kit (D7113) was purchased from Magen Biotech Ltd. (Guangzhou, China). DMF chips were provided by Digifluidic Ltd. (Zhuhai, China).

Several bacterial isolates were identified using 16S rRNA gene sequence analysis. A colony PCR was performed to amplify part of the gene encoding 16S rRNA with primers 8F (5′- AGA GTT TGA TCC TGG CTC AG- 3′, Lane, 1991 ) and 926R (5′- CCG TCA ATT CCT TTR AGT TT- 3′, Amann et al., 1995 (link)) following a program of 96°C for 3 min, 10 cycles of 96°C for 45 s, 50°C for 30 s and 72°C for 2 min, 25 cycles of 96°C for 20 s, 50°C for 30 s and 72°C for 2 min. The 25-μL reaction mixtures contained the template DNA, 5 pmol of each primer (Generi Biotech, Czech Republic), 5 nmol deoxynucleotide triphosphates (dNTPs) (New England Biolabs), 2.5 μg bovine serum albumin (BSA) (New England Biolabs), and 0.5 U Taq DNA polymerase with the corresponding buffer (New England Biolabs). PCR products were purified with the QIAquick PCR purification kit (Qiagen, Germany). Purified products were sent to University of Chicago Research Center DNA Sequencing Facility, USA. Classification was performed by means of Ribosomal Database Project (RDP) Classifier (14) at an 80% confidence threshold.

Restriction enzymes and Deoxynucleotide triphosphates (dNTPs) were procured from New England Biolabs (NEB), United States. All other chemicals and reagents were of the greatest purity and were bought from (Sigma-Aldrich), Mumbai, India and Sisco Research Laboratories. The nickel-nitrilotriacetic acid (Ni-NTA) agarose matrix was acquired from Qiagen, CA, USA. The Himedia Laboratories in Mumbai, India provided the bacterial culture media.