Colorimetric LAMP Kit Biofargo

Input Sample Requirements

• Perform DNA purification with a positive and a negative control. For a sample number of N, perform N+2 purifications. For the two additional purifications，one is for positive control and another negative control.

Storage and Handling

Required Materials not Supplied

Procedural Guidelines

• Perform all steps at room temperature (20–25°C) unless otherwise noted.

• Regularly de-contaminate working areas and equipment using chlorine bleach to avoid potential carryover contamination.

• Avoid opening reaction vessels after a LAMP reaction is completed. But if agarose gel electrophoresis or other post-reaction analysis is desired, perform the analysis in a separated room.

• Screening at least 10 sets of LAMP primers for optimal sensitivity and specificity before choosing a final set.

• Use Loop primers if possible.

• Primers should be HPLC purified.

Procedures

1.  Make 20×Primer Mix. Use appropriate amount of ddH2O to dissolve the HPLC-purified LAMP primers at 100μM each. Use the 100μM stocks to make 100μL 20×Primer Mix according to the following table. If volumes other than 100μL (50μL, 200μL etc.) is desired, adjust volumes accordingly.

1. Thaw all components to be used at room temperature. Vortex briefly 4×Colorimetric LAMP Mix and Template-primer Mix. Centrifuge to collect material and place on ice. Don’t vortex Bst DNA polymerase 2.0.

2. When using the kit for the first time, add all Bst DNA polymerase 2.0 to 4×Colorimetric LAMP Mix and gently invert the tube for one minute to mix thoroughly.

3. For a purification number of N+2, set up N+4 LAMP reactions as described below. Volumes listed are for 20μL reaction. For the two additional reactions, one is for LAMP Positive Control (PC) and another for LAMP Negative Control (NC) .

1. Add 30μL paraffin oil if heat block, water bath is used as incubator. If a PCR machine is used as incubator, turn on the heat lid.

2. Seal reaction vessels.

3. Incubate at 65°C for 60 minutes.

4. Remove tubes or vessels from incubation and examine by eye. Positive reactions will have turned green while negative controls should turn light green. If color difference is not obvious, allow reaction to cool at room temperature.

5. If there is amplification in NC or there is no amplification in PC，the whole experiment is invalid. There is no need to analysis the results of the N+2 samples. Refer to Troubleshooting section.

6. If there is no amplification in NC or there is amplification in PC，the whole experiment is valid. Analyze and record he results of the N+2 samples. The result can be photographed or scanned to record the colorimetric results.

Troubleshooting

1. There is amplification in NC.
   1. This result suggests possible carryover contamination in working environment and/or reagents. To avoid carryover contamination, clean working areas and equipment using a 10% chlorine bleach solution, use new (un-contaminated) reagents, primer stocks, water, etc., use UNG-LAMP Kit, perform experiments in a new (un-contaminated) room.
   2. This result also suggests nonspecific amplification. To avoid nonspecific amplification, do amplification at temperature of 65°C -70°C，put the reaction tubes to 65°C as soon as the reaction is set，use a new set of primer for the targeted DNA region.

2. There is no amplification in PC
   1. This result suggests failures in machine or reagents，use new machine or new set of reagents.

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