Ligation DNA Concentration Calculation Step by step Solution with Explanation

Your question:

A group of students from last year completed ligation under conditions similar to the ones you will be using this week. Refer to the gel picture obtained by this group to fill the summary table provided hereafter. You should assume a ligation molar ratio of insert/plasmid of 4:1 and that you will use 50 ng of plasmid for the ligation. A molar ratio of 4:1 means that there should be 4 molecules of insert for every molecule of plasmid. Your calculations should take into consideration that samples had been diluted with water and the 10X loading buffer before being loaded onto the gel. Assuming that the length of your PCR amplicon and vector are 2583bp and 4333bp respectively, 1)

Ligation DNA Concentration Calculation Answers and Explanation

Analyzing the Ligation Gel Picture

• Insert length: 2583 bp

• Vector length: 4333 bp

1. Calculate Total DNA Needed:

Unfortunately, the information from the gel picture alone isn't sufficient to determine the total DNA concentration needed for your ligation. The gel only shows the presence and relative amounts of different sized DNA fragments, but it doesn't tell us the absolute concentration loaded.

• Since the ligation ratio is 4:1, the fraction of insert molecules is 4/(4+1) = 4/5

Here's the formula to calculate the insert concentration:

Amount of insert required: 1.20 ng