Mixed signal appears in the sequence

Status descriptions

  • There are more than two wave peaks in the beginning.
  • The average signal value is normal.

Possible reasons

  • During the sequencing reaction, other template DNA pollutes it.
  • Primer pollution. Two kinds of primers able to react with the template DNA participated in the sequencing reaction.
  • The PCR product is not fully purified and there are residual forward and reverse primers.
  • Over two sites on the template DNA can be bound by the same primer used for sequencing. It is commonly seen that the PCR product having universal priming site combines to the vector.
  • During the sequencing reaction, the primer annealing temperature is too low.
  • The products of other samples pollute when purifying the sequencing products.

Solutions

  • Make sure that there is only one template DNA. First, make agarose gel electrophoresis to check whether there is pollution of other DNAs, but even though there is only a single product on the gel, it does not mean that trace amount of unexpected products does not exist or there is no other unexpected product in similar size. 
  • Check whether the template DNA can be bound by the same primer used for sequencing.
  • Check whether the residual primers and dNTP are totally removed when purifying the PCR products.
  • Check the Tm value of the sequencing primer. If the Tm value of the primer is over 5℃ higher than the set annealing temperature, please rise the annealing temperature.

 

 
 

Status descriptions

  • There appear over two wave peaks at the same site.
  • The overlapping sequences often appear at the starting site of the cloning site after 50bp, and the lower sequence is different from the expected sequence. The phenomenon is called as two clones.

Possible reasons

  • During the sequencing reaction, other template DNA pollutes it.
  • When selecting the colony, two colonies are selected. This often happen when two single colonies are too close.
  • Make sure that there is only one template DNA. First, make agarose gel electrophoresis to check whether there is pollution of other DNAs, but even through only a single product is seen on the gel, it does not mean that trace amount of unexpected products does not exist or there is no other unexpected product in similar size. 
  • Avoid selecting two colonies at the same time when selecting the colony. In case problems occur, you are recommended to select a new colony.

 

Status descriptions

 The preceding section of the sequence is in disorder and has an obvious end, while the peaks behind are clear to identify.

Possible reasons

Unexpected products appear during the sequencing of PCR products to disturb the sequencing result of main products.

Solutions

Make purification again to remove the unexpected products. If the main products and the unexpected products are in different size, use agarose gel to make separation and then purify to get a single product.