WarmStart Colorimetric LAMP Protocol

Introduction

This protocol comes from the NEB website. We designed 8 sets of LAMP primers to target the MCM7 (KM495430.1) & BT (MG269953.1) fragments of the Ceratocystis fagacearum sequence. The LAMP reaction will be performed using WarmStart (R) colorimetric LAMP 2X Master Mix. For negative reactions, they should result in pink, while for positive reactions, they should result in yellow fragments of the Ceratocystis fagacearum sequence. The LAMP reaction will be performed using WarmStart (R) colorimetric LAMP 2X Master Mix. For negative reactions, they should result in pink, while for positive reactions, they should result in yellow.

Materials

  • Ice box
    • Keep LAMP master mix, the primers and target DNA on ice
  • WarmStart Colorimetric LAMP 2X Master Mix
    • M18000SVIAL
    • Stored at -20 degree celcius
  • Primers
    • DNA templates
      • Nuclease free water
        • Autoclaved PCR tubes (0.2 mL)

          Procedure

          • Prepare 10X Primer Mix containing the LAMP primers
          1. Thaw the F3, B3, FIP, and BIP primers you need.
          1. Vortex each tube briefly (2-3 seconds)
          1. Centrifuge the primers at 3000 RPM for 2 minutes
          1:1:8:8 Primer Master Mix (10X)
          A B C D E F
          1 Primer Starting Conc. Final Conc. (10X) Primer conc. in 25µl LAMP rxn Add this amount of 100µM primer to make primer mix
          2 F3 100µM 2µM 0.2µM 0.5µL
          3 B3 100µM 2µM 0.2µM 0.5µL
          4 FIP 100µM 16µM 1.6µM 4µL
          5 BIP 100µM 16µM 1.6µM 4µL
          6 Water 16µL
          7 Total 25µL <- Add 2.5µL of this to the LAMP Master Mix
          2:2:7:7 Primer Mix
          A B C D E F
          1 Primer Starting Conc. Final Conc. (10X) Primer conc. in 25µl LAMP rxn Add this amount of 100µM primer to make primer mix
          2 F3 100µM 4µM 0.4µM 1µL
          3 B3 100µM 4µM 0.4µM 1µL
          4 FIP 100µM 14µM 1.4µM 3.5µL
          5 BIP 100µM 14µM 1.4µM 3.5µL
          6 Water 16µL
          7 Total 25µL <- Add 2.5µL of this to the LAMP Master Mix
          1. Prepare and label an Autoclaved PCR tube (0.2 mL)
          1. Add 0.5 uL of the 100uM F3 primer stock into the primer mix tube
          • Ask Gary for the P2 pipette. You can use it with a 10uL XL pipette tip
          • Be super slow, much slower than normal pipette speed.
          • And make sure you see that the pipette is taking up some volumes.
          • DON'T HIT IT WITH ANYTHING, this pipette is super fragile and it is much more expensive to calibrate back.
          1. Add 0.5 uL of the 100uM B3 primer stock into the primer mix tube
          1. Add 4 uL of the 100uM FIP primer stock into the primer mix tube
          1. Add 4 uL of the 100uM BIP primer stock into the primer mix tube
          1. Add 16 uL of ddH20 into the primer mix tube. Now you have a primer mix of volume 25 uL.

          LAMP Reaction

          1. Thaw all components to be used at room temperature and place on ice.
          1. Vortex the Master mix for 2-3 seconds
          1. Pipette the Target DNA up and down 10 times very slowly (Pipetting roughly can break the DNA)
          1. Centrifuge the Master mix and Target DNA at 3000 RPM for 2 mins.
          1. Prepare reaction mix in an autoclaved PCR tube as described below using Colorimetric LAMP Master Mix, LAMP primers, and nuclease-free water. The LAMP reaction volume is 25 μl
          LAMP Reaction Mix
          A B C D
          1 Amount for DNA target Amount for no template (negative) control
          2 WarmStart Master Mix 2X (µL) 12.5 12.5
          3 LAMP Primer Mix (µL) 2.5 2.5
          4 Target DNA (µL) 1 <- Add Target DNA Last
          5 ddH2O (µL) 9 10
          6 Total volume 25 25
          1. Vortex reaction mix for 2-3 seconds and centrifuge at 3000 RPM for 2 minutes to collect material.
          1. Check that the reaction solution has a bright pink color, which indicates the initial high pH required for a successful pH-LAMP reaction.
          1. Take a photo of all the tubes at 0 minutes using Gary's camera (Example in template lab notebook entry)
          1. Seal the PCR tubes.
          1. Put the PCR tubes into the PCR thermal cycler. Lock the PCR thermal cycler. Incubate at 65oC for 30 minutes
          00:30:00

          1. Remove tubes or vessels from incubation, put the tube against a white paper, and examine by eye.
          • - Positive reactions will have turned yellow while negative controls should remain pink.
          1. Take a photo of all the tubes at 30 minutes using Gary's camera
          1. If color change is not robust, e.g. an orange color is visible but not obvious, return reactions to 65°C for an additional 10 minutes.
          • If you extended the time, take a photo of all the tubes using Gary's camera
          1. Once the reaction is done, keep it in the 4°C fridge.



          LavaLAMP Protocol using qPCR machine

          Introduction

          We designed 8 sets of LAMP primers to target the MCM7 (KM495430.1) & BT (MG269953.1) fragments of the Ceratocystis fagacearum sequence. The LAMP reaction will be performed using LavaLAMP fluorescence master mix

          Materials

          • Ice box
            • Keep LAMP master mix, the primers and target DNA on ice
          • LavaLAMP Colorimetric LAMP 2X Master Mix
            • Stored at -20 degree celcius
          • Primers
            • DNA templates
              • Nuclease free water
                • Autoclaved PCR tubes (0.2 mL)

                  Procedure

                  • Prepare 10X Primer Mix containing the LAMP primers
                  1. Thaw the F3, B3, FIP, and BIP primers you need.
                  1. Vortex each tube briefly (2-3 seconds)
                  1. Centrifuge the primers at 3000 RPM for 2 minutes
                  2:2:7:7 Primer Mix
                  A B C D E F
                  1 Primer Starting Conc. Final Conc. (10X) Primer conc. in 25µl LAMP rxn Add this amount of 100µM primer to make primer mix
                  2 F3 100µM 4µM 0.4µM 1µL
                  3 B3 100µM 4µM 0.4µM 1µL
                  4 FIP 100µM 14µM 1.4µM 3.5µL
                  5 BIP 100µM 14µM 1.4µM 3.5µL
                  6 Water 16µL
                  7 Total 25µL <- Add 2.5µL of this to the LAMP Master Mix
                  1. Add 1 uL of the 100uM F3 primer stock into the primer mix tube
                  1. Add 1 uL of the 100uM B3 primer stock into the primer mix tube
                  1. Add 3.5 uL of the 100uM FIP primer stock into the primer mix tube
                  1. Add 3.5 uL of the 100uM BIP primer stock into the primer mix tube
                  1. Add 16 uL of ddH20 into the primer mix tube. Now you have a primer mix of volume 25 uL.

                  LAMP Reaction

                  1. Thaw all components to be used at room temperature and place on ice.
                  1. Vortex the Master mix for 2-3 seconds
                  1. Pipette the Target DNA up and down 10 times very slowly (Pipetting roughly can break the DNA)
                  1. Centrifuge the Master mix and Target DNA at 3000 RPM for 2 mins.
                  1. Prepare reaction mix as described below using LAMP Master Mix, LAMP primers, and nuclease-free water. The LAMP reaction volume is 25 μl
                  LAMP Reaction Mix
                  A B C D
                  1 Amount for DNA target Amount for no template (negative) control
                  2 WarmStart Master Mix 2X (µL) 12.5 12.5
                  3 LAMP Primer Mix (µL) 2.5 2.5
                  4 Target DNA (µL) 1 <- Add Target DNA Last
                  5 ddH2O (µL) 8 9
                  6 dye 1 1
                  7 Total volume 25 25
                  1. Vortex reaction mix for 2-3 seconds and centrifuge at 3000 RPM for 2 minutes to collect material.
                  1. Set up qPCR plate
                  1. Put the qPCR plate into the qPCR thermal cycler. Program the device as below.

                  Freeze-dry LAMP reaction protocol

                  Materials

                  For LAMP Reaction

                  For LAMP Reaction

                  Procedure

                  Preliminary steps

                  Turn on freeze-drying machine before sample prep. To wake up the display, tap the screen and enter the password. Press ‘Auto’ on the far left and press start

                  Preparing 10x Primer Mix containing the LAMP primers

                  1. Thaw the PE1 F3, B3, FIP, and BIP primers that you need
                    • Vortex each tube briefly (2-3 seconds)
                      • Centrifuge the PE1 primers at 3,000 rpm for 2 minutes
                        • Make primer mix according to the 1:1:8:8 Primer Mix table.

                          Executing the LAMP reaction on PE1 primers

                          1. Thaw all of the LAMP reagents needed at RT and place on ice immediately
                            • Vortex each tube briefly (2-3 seconds)
                              • Pipette the target DNA up and down (10 times very slowly; make sure not to do it too roughly as it can break the DNA)
                                • Centrifuge the master mix and target DNA at 3,000 rpm for 2 minutes
                                  • Prepare reaction mix as described in the LAMP Reaction Mix table, but replacing Warmstart Master Mix with LavaLAMP Master Mix.
                                    • Vortex reaction mix for 2-3 seconds and centrifuge at 3,000 rpm for 2 minutes to collect all materials

                                      Freeze-drying PE1 LAMP primers

                                      1. Using a fresh, large bore needle, carefully poke 2 holes into the tips of the lids and be sure to remove any plastic that might have fallen into the tube
                                        • Transfer sample into aerated tubes
                                          • On a clear surface, lay out the aluminum and place the paper towel on top
                                            • Place the acrylic plate on top of the liquid nitrogen and wait for it to stop aggressively bubbling
                                              • Using tongs, carefully place the tubes into the liquid nitrogen ensuring the liquid freezes at the bottom of the tube/well
                                                • Release the tubes into the liquid nitrogen
                                                  • Once fully frozen, use tongs to take out the acrylic plate, over the white container with a few gentle shakes to ensure all the liquid nitrogen is gone and place it on the paper towel that is overtop the sheet of aluminum foil
                                                    • Take out tubes and place on top of the frozen acrylic plate. MAKE SURE TO GIVE THE TUBES A SHAKE TO ENSURE NO EXCESS LIQUID NITROGEN IS PRESENT
                                                      • Carefully wrap the tubes up and place in glass freeze fry container and attach the lid securely. Inert the container into one of the openings with gentle but firm pressure. Ensure all the reading on the machine are in green. Turn the balck knob to the right so that the bevealed edge is in line with the metal attachment on the lid of the glass bottle, you should hear a hissing sound
                                                        • Wait until all the values drop back down on the machine and let run overnight
                                                          • In the morning, close the nozzle by turning the knob to the left. Match its position with the other nozzles on the machine
                                                            • Turn off the vacuum and collector on the display
                                                              • Press the power button to put the machine into standby mode
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