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Learning Module - Experiment 3C

Estimation of DNA yield from phage and E. coli XL-1 Blue

Your gel picture should look something like this:

gel with bands

Do not make any marks on the picture itself. After affixing the picture in your notebook, proceed to add labels and a legend.

Each lane should be numbered from left to right (with the sample wells at the top) starting with Lane 1. Each major band should be identified with a letter and a Legend describing each lane must be placed below or next to the picture:

Labled gel with bands

By comparison of fluorescence intensities, estimate the amount of DNA in each of your preparations:

For pTrc99A, Lane 4 (2 μL) has about the same amount of DNA as the Standard in Lane 3 (200 ng) and Lane 5 (4 mL) has about twice as much as Lane 3.
Lane 6 contains too much DNA to be able to estimate accurately by comparison to the standards, although it looks to be about 3 times as much as Lane 3.

Therefore, the concentration in the mini-prep is: 200 ng / 2 μL = 100 ng / μL, or (2 x 200 ng) / 4 μL = 100 ng / μL
and the yield is: 100 ng / μL x 30 μL = 3000 ng, or 3 μg

For ZAP/topAcysB, Lane 10 (2 μL) has about the same amount of DNA as Lane 7 (50 ng): 50 ng / 2 μL = 25 ng / μL
and Lane 11 (4 μL) has about 3/4 the amount of DNA as in Lane 8 (100 ng): (100 ng x 0.75) / 4 μL = 18.75 ng / μL
Therefore the concentration in the mini-prep is: (25 ng / μL + 18.75 ng / μL) / 2 = 21.9 ng / μL
and the yield is: 21.9 ng / μL x 60 μL = 1313 ng or 1.31 μg

From the concentrations, the volume of each mini-prep needed for use in the subcloning (Experiment 4B) can be calculated, and from the yields, a comparison of the actual yield to the theoretical DNA yields can be made. If one assumes 100% recovery of both DNAs, the % viability of the phage can be calculated and the copy number of pTrc99A can be determined. These topics should be addressed in the Discussion section for Period 3C.