Diagnostic Digest Assignment PDF

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Diagnostic digest worksheet...

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Diagnostic Digest Assignment Please utilize the results from the diagnostic digest that show data from 3 different groups in your class. Please use the background provided to complete this assignment. With the results provided from the 3 groups, I expect you to analyze this data and write up a discussion. For each group provided I expect the following in the discussion: 1. A summary of the data from each group (please include all bands observed from each restriction digest) 2. A statement as to if the experiment worked for each group o If you think it did, how does the data support it o If you think it didn’t, how does the data support it o If you think it kind of worked, how does the data support this 3. A conclusion statement that includes why you think the experiment worked/didn’t work for each group. What may have gone wrong or right. Cloning Background: In lab you are trying to clone a PCR product into the L4440 plasmid utilizing the KpnI and XbaI restriction enzyme recognition sites present in the plasmid as well as the PCR product. After restriction digestion, gel purification, ligation and transformation it is possible that the vector does not take up the insert. To verify that the cloning experiment has worked and our target gene has been incorporated into the L4440 vector, we need to isolate the plasmid DNA from the bacteria and perform a diagnostic restriction digest. In order to generate enough plasmid to be able to analyze on an agarose gel we will need to pick individual colonies and grow the cultures overnight prior to plasmid purification. Another way to identify colonies that contain the PCR product (insert) is to take advantage of Blue-White screening. Unfortunately the vector that we are using does not allow for this. In order to verify the piece of DNA has been successfully cloned into the vector that is being used a diagnostic restriction enzyme digest can be performed. This takes advantage of the fact that when restriction enzymes are used that are known to cleave at specific places within the cloned plasmid a certain banding pattern can be expected on an agarose gel. Often, the size of the plasmid insert and vector backbone are known and thus this technique can be quickly used to verify your plasmid. The goal of a diagnostic digest is to cut your plasmid into specific sized pieces and analyze the resulting fragments by gel electrophoresis. The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert. By selecting the appropriate enzyme(s), one can either linearize a plasmid to determine the size of the entire construct or excise some or all of an insert from it. To determine the size of restriction fragments, the fragments in a digest are separated by electrophoresis in an agarose gel that also includes DNA molecules of known size as standards (DNA ladder).

Figure 1. Empty L4440 vector and vector containing PCR product of gene of interest utilizing XbaI and KpnI restriction enzymes for cloning.

The L4440 plasmid is 3kb and the PCR insert is 1kb. If the plasmid takes up this insert and a double digest is performed with KpnI and XbaI 2 bands would be expected after running and agarose gel. The vector band at 3kb and the insert band at 1kb (Figure 2).

Figure 2. Expected results from digest with KpnI and XbaI

For the diagnostic digest being performed, it is important that the digestion of the DNA be complete. When a digest is complete, the resulting fragments should be present in equimolar quantities, but the amount of mass of each fragment will vary, depending on its size. The ethidium bromide fluorescence is proportional to the size of the fragment. Therefore, when looking at a digest, the intensity of each band should decrease as you proceed from the largest fragments to the smallest. If a band appears lighter than its size would suggest, it could be the product of a partial digest. If a band appears brighter than expected, it could be the result of

two separate fragments from different regions of the plasmid that are essentially the same size (a doublet). It is important to be able to recognize these patterns in gels when restriction mapping (Figure. 3).

Figure 3. Results showing restriction enzyme digest of empty vector, vector containg insert, vector containg insert showing a partial digest, empty vector and digest with no staining in lanes 1, 2, 3, 4 and 5 respectively.

Diagnostic Digest Results: Group 1

Group 2

Group 3...