Question

After PCR is performed the products are run out on an agarose gel. In the figure below, grey bands represent the wells the PCR product was loaded into. The white bands represent DNA fragments produced by PCR. The target fragment amplified by the primers was 1,500 bp in size.

The ladder is a standard DNA ladder containing bands of various sizes between 5,000 and 1,000 bp. The negative control contained only molecular grade water*. The positive control contained DNA known to contain the 1,500 bp target. Samples 1-3 are random environmental samples.

Answer the following questions using the word bank provided below-

-Based on the observed banding pattern, what went wrong?

-To correct the problem, the MgCl₂ concentration could be:

or

-the annealing temperature could be:

-Which samples likely contain the target DNA sequence?

Word bank:

non-specific binding

failure to anneal to the target DNA

contamination

increased

decreased

sample 1

sample 2

sample 3

samples 1, 3

samples 1, 2, 3

further work must be done to assess which samples contain the target sequence

5,000 bp 4,000 bp 3,000 bp 2,000 bp 1,000 bp

Answer 1

No bands can be observed in the lanes of control and sample. The only lane which shows bands is the ladder.

I suggest that the PCR did not work for any of the samples and hence no banding was observed. MgCl2 aids is maintaining the pH of the reaction mixture which also helps in enzymatic action of the DNA polymerase.

I suggest performing the PCR again with lesser quantity of template DNA. That would help and you will get some results.