Content Status
Type
Linked Node
PCR Reagent Preparation Procedure in the LPA Lab
Learning ObjectivesPCR Reagent Preparation Procedure in the LPA Lab
H5Content
Content
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.
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.
Kindly provide your valuable feedback on the page to the link provided HERE
LMS Page Link
Content Creator
Reviewer
- Log in to post comments