How to perform an MLPA reaction | by MRC-Holland
Hello, Welcome to this instruction video on how to perform an MLPA reaction. Before you start make sure that you use the latest version of the MLPA protocol provided by MRC-Holland, as all probemixes are optimized to work with this protocol. Take the DNA samples, SALSA MLPA probemix and SALSA MLPA buffer needed for day 1. And write down the lot numbers. Then start by pipetting the DNA samples. Mix the samples by vortexing briefly and always spin down shortly afterwards, as drops may have adhered to the lid. Do this for all samples. Mark PCR tubes and caps. and pipette 5 microliters of DNA sample with a concentration of 10-20 nanograms per microliter to each tube. Place the tubes in the thermocycler. Make sure to use a calibrated thermocycler with a heated lid to reduce evaporation. Start the MLPA programme. First denature the DNA samples at 98 degrees for 5 minutes and afterwards keep the samples at 25 degrees. Meanwhile prepare the hybridisation master mix. Prepare master mixes to simplify pipetting and to reduce variation between samples. Mix the probemix and MLPA buffer before use. Vortex and spin. Prepare larger volumes of master mix solutions than needed. An extra 5-10% volume ensures you do not run out of master mix while adding it to the samples. Pipet per reaction 1.5 microliters MLPA probemix and 1.5 microliters MLPA buffer. Again mix by a brief vortex and spin. After denaturation, the thermocycler will cool down to 25 degrees. Open the tubes. If you use cap strips start in the middle of the strip to prevent possible contamination due to sample spattering. Add 3 microliters of hybridisation master mix to each sample. Mix gently by pipetting up and down. Close the tubes properly to prevent evaporation. But be careful not to push too hard as this might deform the tubes. Continue the programme to step 3. Denature the samples again for 1 minute at 95 degrees. Then the programme will continue to step 4: 60 degrees. Hybridise the DNA samples overnight for 16-20 hours at 60 degrees. This is the end of day 1 of the MLPA procedure. Continue on day 2. Take SALSA ligase buffers A and B and SALSA ligase-65 enzyme needed for the ligation reaction. Mix the ligase buffer A and buffer B before use. Vortex and spin. Never vortex the enzymes. Prepare the ligase master mix at room temperature right before the ligation reaction. Start with the largest volume and add the enzyme last. Pipet per reaction 25 microliters ultrapure water, 3 microliters ligase buffer A, and 3 microliters ligase buffer B, and mix by a brief vortex and spin. Then warm the ligase-65 enzyme in your hand for 10 seconds to reduce viscosity. Per reaction add 1 microliter ligase-65 enzyme to the ligase master mix. Check if there are no droplets of enzyme on the outside of the pipette tip. Mix the master mix by slowly pipetting up and down. Use a pipet tip that is sufficiently large to allow proper mixing. Note that the ligase master mix must be at room temperature before adding it to the samples. This will prevent the formation of non-specific peaks. Distribute the ligase master mix to PCR tubes for multichannel use. This is essential to limit the time that the tubes are left open at 54 degrees, thus minimizing evaporation. Continue the programme to step 5: 54 degrees. Open one strip or row at a time. And add 32 microliters ligase master mix to each sample when the samples are at 54 degrees. Check the pipette tips to see if the volume is equal in all tips. Mix gently by pipetting up and down. Try to prevent the formation of air bubbles by emptying the pipette tip slowly on the side of the tubes. Check if all pipette tips are empty. If there are any air bubbles present at the bottom of the tube, quickly tab the tubes on the thermocycler or table. Continue the programme to step 6 for ligation:
15 minutes at 54 degrees. After 15 minutes of ligation the programme will continue to step 7. In this step the ligase-65 enzyme will be inactivated for 5 minutes at 98 degrees, afterwards keep the samples at 20 degrees. During this time you can take SALSA PCR primer mix and SALSA polymerase enzyme for the preparation of the PCR. Mix the PCR primer mix before use, vortex and spin. Vortex and spin. Prepare the polymerase master mix at room temperature right before the PCR. Pipet per reaction 7.5 microliters ultrapure water – and 2 microliters PCR primer mix. Again mix by a brief vortex and spin. Warm the polymerase enzyme in your hand for 10 seconds and add per reaction 0.5 microliters polymerase to the polymerase master mix. Mix by slowly pipetting up and down until the master mix is properly mixed. Distribute the polymerase master mix to PCR tubes for multichannel use. At room temperature, add 10 microliters polymerase master mix to each sample. Mix gently by pipetting up and down Close the PCR tubes and put them back into the thermocycler. Continue the MLPA programme to step 9.
Amplification in 35 cycles of: 30 seconds at 95 degrees, 30 seconds at 60 degrees and 60 seconds at 72 degrees. End with 72 degrees for 20 minutes. and afterwards keep the samples at 15 degrees. This is the end of the MLPA reaction. Take the tubes to your sequencing facility or store them at 4 degrees for a maximum of 1 week. Make sure to wrap the PCR products in aluminium foil as fluorescent dyes are light-sensitive. Thank you for joining us in this video. Want to learn more? Subscribe to our
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