Antibiotic Resistance: Methods and Protocols by Stephen H. Gillespie

By Stephen H. Gillespie

Scientific and diagnostic microbiologists aspect molecular and actual equipment for learning the becoming challenge of antibiotic resistance in micro organism, and facilitate new antibiotic study courses to aid redress the matter. The concepts diversity from those who supply quick analysis via DNA amplifications and phage demonstrate, to these for plotting the transmission of resistant organisms and investigating their epidemiology. in addition to illuminating the elemental biology of antimicrobial resistance, additionally they advance and enforce now diagnostic and epidemiological instruments.

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1% N-lauryl sarcosine. Gentle heating may be required to dissolve the guanidine thiocyanate. This solution is stable at room temperature, however precipitates may form in cool rooms. Precipitates redissolve rapidly with gentle heating. 13. A water bath set at 37°C. 14. A heating block set at 85°C. 15. 6 using NaOH. 16. 1-methyl-2-pyrrolidinone (Aldrich Chemical, Milwaukee, WI). Warning: corrosive. 17. Phenol-Water-Chloroform (Applied Biosystems, catalog #400765). Warning: corrosive. 18. Chloroform.

The deposit is heated to 80°C for 20 min in a waterbath. 100 µL of chloroform is added and the tube vortexed for 30 s. The sample is microcentrifuged (12,000g for 1 min) and the aqueous layer used as the PCR sample. 4. Samples must be heat killed (80°C for a minimum of 20 min) before leaving the Category III (P3) facility. 36 McHugh 5. This method of DNA preparation yields DNA in adequate quantity and quality for PCR. As this DNA preparation is not pure it is likely to deteriorate rapidly on storage.

8. Check the quality of the PCR MIMIC DNA by gel electrophoresis. 5. QUANTIFICATION OF MTB-SPECIFIC PCR MIMIC The quantity of the PCR MIMIC can be determined by comparing the intensity of the electrophoretic bands produced by the PCR MIMIC with the intensity of bands generated by known quantities of qX174/Hae III size markers, provided in the PCR MIMIC™ Construction kit. 1. Set up three dilutions of qX174/Hae III digest size markers (100 ng, 200 ng, and 400 ng) and one dilution of the purified Mtb-specific PCR MIMIC DNA (1 µL of the total purified products) using sterile H2O, 10X PCR buffer, and 6X dye in a final volume of 15 µL.

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Antibiotic Resistance: Methods and Protocols by Stephen H. Gillespie
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