Help me 1 and 2 and 3 Understanding your actions 1. Assume we used a different p

Question

Help me 1 and 2 and 3

Understanding your actions 1. Assume we used a different pair of PCR primers that contained a higher percentage C content. Naturally, these primers would still surround the LEU2 locus but would bind to other bases upstream and downstream of the gene. Explain how and why uration, annealing, extension). 2. If your results next week reveal a replicated product in only one of your samples, what is not the reason the other sample failed to amplify? Explain your answer We have used EDTA in the past to protect genomic DNA from enzymatic degradation by DNAses in the cell. Why did we not use this reagent to protect our template during the long PCR procedure? 3.

Explanation / Answer

1. Changing the primers with ones that have a bigger concentration in GCs will influence mainly the annealing and the extension steps of the PCR. The first one because we would need to standardize again the temperature needed for annealing, it would not be 52°C anymore because this was based on the original concentration of GCs in the primers. The extension time could also change: assuming that the primers anneal in upstream or downstream regions of the target gene, then it would take more time to copy the whole target and the extension time would then be longer. There would be no change for the de-naturalization step because the temperature needed doesn´t need to be different when the amount of GCs or ATs differ.

2. that there was succesful homolougus recombination, because even if there was there would be an amplified product of the PCR but with different size than the not recomined sample.

3. Because during the PCR procedure, you do not have the cells products anymore, so you do not have the DNAses. On the other hand, EDTA can interfere with the PCR by capturing the Mg2+ ions, which is one if the co-factors needed to amplify the DNA, interrupting the PCR procedure.

Related Questions

Navigate

• Browse (All)
• Browse H
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.