Oct 30, 2024
Version 1
meat identification protocol using LAMP V.1
This protocol is a draft, published without a DOI.
- 1OBS
Protocol Citation: openbioscience Adrian 2024. meat identification protocol using LAMP. Protocol exchange https://protocols.io/view/meat-identification-protocol-using-lamp-dqur5wv6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 29, 2024
Last Modified: October 30, 2024
Protocol Integer ID: 111217
Abstract
Identifying species by identifying unique DNA sequences is common practice.
In this case we will identify the presence or absence of pork DNA in a meat sample.
We will use the alkaline method for DNA extraction and LAMP method for DNA amplification and detection.
This is not a new protocol but rather the detailing of a previously published protocol listed in the manuscript citation.
Materials
Materials
Consumables: minimum 2 PCR tube2 0.1 mL PCR tubes with flat caps, commonly known as low-profile PCR tubes that matches the device well size to store the ready to be tested sample (aliquot).
Labware: graduated cylinder for measuring water
Devices:, LAMP/PCR/Water bath and thermometer, scale, pH meter if using Tris, 1- 10 μl micropipette, 100- 1000 μl micropipette and tips, centrifuge, Optional: vortex, scale and boat.
Reagents: Primers, LAMP master-mix, DNase free water, aliquot of meat to be tested in a prepared form
Others: nitrile gloves, pipette rack , tube rack, labeling pen
Safety warnings
Use appropriate PPE, follow Safe Work Practices and follow local regulation.
prepare 100 μM primer stock for each of the 4 primers F3 B3 FIP BIP. Divide into several aliquots and store at -15 Celsius
centrifuge the lyophilized primer tubes to move the powder to bottom as per IDT guide
Add 264 μL DNase free water to F3, 242 μl to B3 tube, 277 μl to FIP tube and 271 μl to BIP tube. Note: water can be substituted with 10 mM Tris pH8.0 (25 Celsius). Note the quantities for 100 μM were specified by IDP in "oligonucleotide specification sheet" on their product.
vortex all 4 primer stock tubes
divide in small aliquots of maybe 10 μl to minimize freeze thaw cycle
store at -15 to -20 Celsius for max 2 years
prepare ice tray for the reaction
thaw primer stock aliquot and superscript LAMP master mix.
label "target" 100 μL tube
label NT (no template negative control) tube
on ice tray add 0.2 μM F3 primer to the target tube and NT tube. As final volume is 25 μL and stock concentration is 100uM, that means (25μl x 0.2μM)/100μM = 0.05 μl F3 primer.
Note: Detailed explanation of the calculation at https://www.optigene.co.uk/wp-content/uploads/2012/06/OptiGene-LAMP-User-Guide-Assay-Design-Primers-1.pdf
on ice tray add 0.2 μM B3 primer to target tube and NT tube. As final volume is 25 μL and stock concentration is 100uM, that means (25μl x 0.2μM)/100μM = 0.05 μl B3 primer.
on ice tray add 0.8 μM FIP primer to target tube and NT tube. As final volume is 25 μL and stock concentration is 100μM, that means (25μl x 0.8μM)/100μM = 0.2 μl FIP primer.
on ice tray add 0.8 μM BIP primer to target tube and NT tube. As final volume is 25 μL and stock concentration is 100uM, that means (25μl x 0.8μM)/100μM = 0.2 μl BIP primer.
on ice tray add ? μL at ?X concentration TODO of tubes from https://www.fishersci.ca/shop/products/superscript-iv-rt-lamp-master-mix-3/p-9568005 mix to target tube and NT tube
add 1 μL template DNA to target tube and 1 μL DNase free water to NT tube
add DNase free water to 25 μL to both target and NT tube
load target and negative control in the LAMP device and perform the LAMP protocol as specified by manufacturer
Protocol references
2 Girish et al: Rapid detection of pork using alkaline lysis- Loop Mediated Isothermal Amplification (AL-LAMP) technique. Published, journal pre-proof can be downloaded from Academia https://www.academia.edu/114013841/Rapid_detection_of_pork_using_alkaline_lysis_Loop_Mediated_Isothermal_Amplification_AL_LAMP_technique