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1. Pipetting probemix and MLPA buffer.
   • Small pipetting errors, resulting in a reduced amount of MLPA buffer in one or more reactions, can result in higher signals of certain probes.
2. DNA samples should contain at least 5 mM Tris buffer.
   • Use TE (not dH₂O) to dilute concentrated DNA samples. Unbuffered DNA samples are rapidly degraded by depurination in the initial 5 minute 98 °C DNA denaturation treatment.
   • In case it is not known whether the DNA samples are dissolved in TE or in water, add 10 mM Tris-HCl pH 8.0 to each sample.
   • For more info see www.mlpa.com → MLPA procedure → sample treatment.
3. Take care of sample quality.
   • The sample A260/280 ratio is not a good indication of its suitability for MLPA, as sample contamination with proteins does not influence your MLPA results.
   • The presence of salt (>40 mM) can cause false positive results as it prevents the complete denaturation of CG rich areas of the chromosomes.
   • Other impurities which can influence MLPA include substances like heparin and melanin that are present in many tissues. Not all purification methods are suitable to remove these substances. Whenever possible, compare only DNA samples that were purified by the same method.
   • Do not concentrate diluted DNA preparations by Speedvac as it may result in a very high EDTA concentration, which influences the MLPA reaction.
4. Add polymerase mix at room temperature, not at 60 °C.
   • The new protocol, introduced in 2011, requires a PCR reaction setup at room temperature.
5. Use 50-200 ng of sample DNA.
   • Using more than 200 ng of sample DNA can reduce the quality of the results. The Q-fragments at 64-70-76-82 nt indicate whether or not sufficient amount of sample DNA is used.
   • Use approximately the same amount of DNA for all samples within one experiment.
6. Use at least 3 reference samples in each experiment to enable reliable data normalisation..
   • The use of multiple reference samples increases the confidence of your normalisation and can aid in result interpretation, as well as in troubleshooting.
7. Check reproducibility of probes in your MLPA experiments!
   • Standard deviation of all reference probes in the samples tested should preferentially be below 0.10.
   • Standard deviation of all probes in the reference samples should preferentially be below 0.10.
8. Do not use old capillaries or deteriorated gel..
   • Old capillaries and/or deteriorated gel will cause unreliable results.
9. Take care that the MLPA peaks are within the optimal range of the CE device..
   • Abnormal results can be obtained when peaks are too high or too low. Each CE device has its optimal signal intensity range, which can be found in the General MLPA Protocol.
10. Confirm MLPA results.
    • Confirm results by another technique or by an MLPA probemix with more probes in that gene or chromosomal region.
    • False positive duplications can e.g. be the result of small pipetting errors of the MLPA buffer (see point 1).
    • False negative deletions can e.g. be the result of a polymorphism or point mutation in the sequence detected by the probe, sometimes even 10-20 nt from the probe ligation site.
11. Follow the General MLPA Protocol and read the product description..
    • For correct interpretation of results, a good knowledge of the technique and of the specific product used is essential.
    • Do not modify the protocol provided by MRC-Holland. All our products are optimised using this protocol, which is carefully chosen to minimise experimental variation.