Colony PCR is performed to confirm whether the insert gene has been successfully inserted into the target vector. The primer sequence design can involve primers on both strands specific to the insert fragment, primers specific to the vector, or one primer on the insert fragment and the other on the vector. Different design strategies will lead to different interpretations during further analysis..
Here are simple drawings of three different design plans. (The insert gene is 500 base pairs long.)

Fig. A: Primers on both strands are located on the insert fragment.
Fig. B: Primers are designed on the vector, with distances of 132bp and 56bp from the insert gene insertion site, respectively.
Fig. C: One primer is on the insert fragment, and the other is on the vector, with a distance of 56bp between the primer on the vector and the insert gene insertion site.

Question 1： If the primer design strategies shown in Fig. A, B, and C are respectively used, would colony PCR produce products if the insert gene is successfully inserted into the vector? If so, how large would the products be? Please provide the results of insert success and failure for all three strategies. If there are products, draw the predicted positions and indicate the sizes next to the DNA marker. (If there is no product after PCR，just draw ‘X’ on the answer box) (60%, one answer for 5%.)

Question 2：The primer design strategy for this semester corresponds to which figure? (5%)




















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Winny wants to obtain the pET28b-eGFP plasmid today. Firstly, she uses restriction enzymes to cut the required pET-28b vector and eGFP gene. Then, she ligates them together and transforms them into E. coli. After selecting the grown colonies, she performs colony PCR and gets a product size of around 1000bp, which matches the predicted size.

Question: Does this mean the colonies Winny picked truly contain the target eGFP gene? What concerns might arise? If she wants to confirm whether she has indeed selected colonies containing the pET28b-eGFP plasmid, what methods can she use? (Hint: from last semester's course)(35%)