PCR Reagent Preparation Procedure in the LPA Lab

In a Line Probe Assay (LPA) lab, subsequent to DNA extraction, reagents are prepared for PCR amplification using the checklist and worksheets.

Preparation Steps

• Prepare a written checklist of materials and tools needed.
• Prepare a worksheet with a list of specimens (identifiers) to be tested.
• Prepare worksheet to calculate the volume master mix ingredients (put on the wall near work area). ​

Setting up the Workplace

• Put on gloves and lab coat.
• Decontaminate biosafety cabinet and work area (1% Sodium hypochlorite solution, followed by 70% alcohol​).
• Cover work area in a biosafety cabinet with clean paper towels.

Calculations for Master Mix Preparation

• Determine the number of specimens and controls.
• Use this number “+” 1 to calculate the volume of different components of the master mix.
• The volume of the master mix is 45 μl per specimen: ​
  • 10 μl AM-A
  • 35 μl AM-B
• Prepare the number of tubes required.

Polymerase Chain Reaction (PCR) Reagent Preparation Procedure in Master Mix Room

• Reagent preparation for both First Line - Line Probe Assay (FL-LPA) and Second Line - Line Probe Assay (SL-LPA) remains the same with only difference in the kits used. The master mix must be freshly prepared.

Click the video below to see how to prepare the Master Mix.

https://empowerschoolofhealth-my.sharepoint.com/:v:/g/personal/admin_empowerschoolofhealth_org/EfqikUtgzDlKrZpxwoLdMGMB0Oyc_-LiPP8Z7zaQKmZkKQ?e=ElmNpH

Resources

• GenoType MTBDR plus ver 2.0 Kit, Instructions for Use.
• GenoType MTBDRsl VER 2.0 Kit, Instructions for Use.
• GLI Training Package on LPA.

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