The problem is such:

After performing a PCR, the vector carrying the PCR fragment with two restriction enzymes (Nhe1 and Asc1). The DNA samples were then separated using agrose gel electrophoresis shown below: enter image description here

It is given that the PCR product is 3.2kb in the figure below: enter image description here

The question now asks:

  1. Which DNA fragments do not have the expected sizes?

  2. How could you explain the DNA fragments with unexpected sizes?

  3. How could this problem be avoided?

My thinking is that the fragments in lane 3 do not have expected sizes as with only double digestion one fragment should be close to the undigested product in lane 2 and not two...but I'm not sure if this is right. I know that the Nhe1 and Asc1 restriction sites are at the ends of PCR product so there should not be two bands which can add together to give the original...am I making sense? In addition, could the temperature be increased to reduce non-specific primer annealing?

Can please someone please guide me along the right lines by explaining their reasoning and offer any advice on how unexpected bands can be avoided? Thanks!

  • $\begingroup$ +1 for a decent homework question, but I find neither the recognition sites of NheI nor AscI in either the 5' or 3' DNA sequences. $\endgroup$ – March Ho Jan 3 '16 at 2:17
  • $\begingroup$ @MarchHo i have designed my own primers in a previous section. I included restriction sites at the 5' end of the forward and reverse primers $\endgroup$ – justbehappy Jan 3 '16 at 9:44

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