Question

Question 1. 1 2 3 4 5 6 7 8 Lane Name/Code Ladder 1B | 2B 2 DNA Sample/Treatment DNA ladder Digest Digest Digest Digest Digest Digest 3B 4B 5B Negative Figure 1: 1% Super buffer agarose gel electrophoresis of restriction digest of the plasmid containing the gdhA gene insert. Based on the information above answer the following questions? 1. What ladder size used? 2. What are the two top bands and bottom bands representing? 3. Explain why the observed bands were located above the ladder? How can we improve the result? 4. If it is possible, what was the approximate size of the observed PCR products on the agarose gel? What was the size of the expected PCR product? 5. Is there any evidence of primer dimers on the agarose gel? Explain your answer. 6. Any evidence of non-specific amplification for any of the test sample ID numbers (i.e. smearing or multiple bands). If so, which samples? 7. Comment/conclude on the agarose gel results?

Answer 1

Ans.1- Ladder size is only determine when the plasmid size is known. And in this question plasmid size is not given. Please provide plasmid size if wants to know ladder size.

Ans.2- Top bands representing plasmid and bottom bands representing gene of interest.

Ans.3- Observed bands were located above the ladder because observed bands contains plasmid which size is larger than ladder size. We can improve the result by changing the ladder of greater size range.

Ans.4- If we don't know the size of plasmid and ladder we cannot estimate the size of observed PCR product and expected PCR product. In this question size of plasmid and ladder are not given.

Ans.5- No any evidence of primer dimers on this agarose gel because primer dimers are of very small in size and run faster than ladder and if primer dimers are present in this gel then bands of primer dimers should be present at the bottom.

Ans.6- Because it is restriction digestion then there is no any evidence of non- specific amplification for any of the test sample ID.