2. You ran your PCR with the following machine parameters: 94°C for 1min, 55°C f

Question

2. You ran your PCR with the following machine parameters: 94°C for 1min, 55°C for 30sec, 72°C for Imin. You expected a PCR product of 3Kb but saw nothing on in the lane on the agarose gel. What should you change to get the product you want? 3. If you started with 10 copies of a DNA fragment and you ran a PCR for 5 cycles, how many copies of the DNA would you have at the end of the run? 4. The forward primer for your PCR has this sequence: 5'-TACGGTTAACGCGTATGCTG-3'. What is the Tm for this primer? 5. An agarose gel shows that your PCR of a chipmunk gene succeeded in producing a product of 2Kb which is what you expected. You clone and sequence the PCR DNA product. The result of a computer analysis show that the DNA that was sequenced was human and not chipmunk. What went wrong? What could you do to look out for this problem?

Explanation / Answer

Answer

2. Annealing and extension temperatures are not optimal.

Annealing temperature = lowest primer Tm - 5 °C, Extension temperature = 72 °C.
Decrease annealing temperature by 6 to 10°C.

3. 320 copies of DNA fragment.

4. Tm = 2 X (A+T) + 4 X (G+C)
      = 2 X (4+6) + 4 X (6+4)
      = 2 X 10 + 4 X 10
      = 20 + 40
      = 60 C

5. Primer design went wrong. It primer is match with the human DNA sequence not the chipmunk.

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