After PCR amplification, agarose gel electrophoresis analysis is generally carried out. After electrophoresis, there are generally the following bands:
1, primer band. When the concentration of the primer is too high or the amplification efficiency is not particularly high, it is generally not present as a fine band and is divergent; if the target amplification product band and the primer band are both bright, it is considered to appropriately reduce the amount of the primer.
2. Primer dimer band. It runs slower than the primer, and the band is clear, but if the amplification product is less than 100 bp, the product and the primer dimer are not good. It is recommended to use DNA polyacrylamide electrophoresis (not SDS polyacrylamide electrophoresis of protein); If the primer dimer still seriously affects the amplification of the target product after optimizing the refolding temperature, it is preferred to replace the primer design (because the cost of primer synthesis is lower than the cost of repeated optimization conditions)
3. The target amplification product band. The size is the same as the design size and the strips are clear.
4. Non-specific amplification product bands. The size is not the same as the design size, and the strips are clear. It is generally considered to reduce or eliminate by increasing the tempering temperature (the temperature gradient function can also be used to find the optimum tempering temperature, the Dongshenglong 811 type PCR instrument has this function). If the fragment size is larger than the target fragment, it can be reduced or eliminated by reducing the extension time (ie, not giving it enough extension time). Another non-specific amplification product band is the contamination of genomic DNA from reverse transcription PCR experiments. If there is an intron in the amplification product of interest, the amplification product is likely to be larger than the target gene. This band cannot be eliminated by optimizing the renaturation temperature and can be sampled with RNase-free DNase prior to reverse transcription.
5. Template DNA bands. It may appear when the template concentration is too high. If genomic DNA is used as a template, the bands may be messy and large. Generally, when performing PCR amplification, the concentration of the template should not be too large, otherwise some impurity DNA (may not be banded or seen) longer than the target gene will be produced, which is caused by the principle of PCR amplification.
If you have any questions, you can go to Dongsheng Innovation Baidu Post Bar "PCR technology and PCR instrument bar" to ask questions, will answer for you in detail. Or follow my official WeChat "eastwin_long" or "eastwin_mi".
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