Question

4. 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 30 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 Estimate the concentration of the purified digested insert and plasmid (gel below). 2] Using the concentrations obtained in 1), fill the table with the appropriate volume of the different components of the ligation reaction(see Appendix D4). Please include all your calculations. Read the question carefully since last year ligation was a bit different. In

Answer 1

Lane 2. Pcr digested amplicon ... the band intesity is approximately 1.5 times of the represenattive band of the size 2000bp. The band of the amplicon is in between 2500 and 3000, it means the band of the marker is 30 ng for 2500 bp therefore for 2583 bp it should be 30X2583/2500=30.996 and the band intesity of the amplicon is 1.5 times, it means 30.996X1.5=46.5 ng/ul

lane 3. The size of vector is 4333 and the band concentration at 4000 is 40 ng it means for 4333 the concentration should be 40X4333/4000= 65ng/ul but the band intensity is approximately 0.7 times, it means the actual concentration of vector is 65X0.7=45.5

for 4.4 kb of vector 30 ng is taken for ligation it means 30/45.5=0.659=0.7 ul of plasmid should be taken for the ligation

for insert:vector molar ratio of 4, insert should be taken according to the vector.

30ng of vector is taken for its size of 4.4 kb it means 30/4.4=6.8=7 ng/kb vector is taken, therefore for 4:1 ratio of insert and vector, insert should be taken 7x4=28 ng/kb

it means 28x2.583=72 ng of insert shoul be taken. insert concentartion according to the gel is 46.5 ng/ul

72/46.5=1.55 ul

finally

Vector=0.7ul

insert=1.55

5X buffer= 8 ul

enzyme=2ul

water=27.75

total= 40 ul