The endonuclease Ceu-I recognizes a 19 bp sequence. How often would you expect this enzyme to cut the 4.8 x 106 bp chromosome of Salmonella typhimurium?
The chromosomes of many enteric bacteria, including Salmonella typhimurium, Escherichia coli, and Klebsiella aerogenes have been digested with Ceu-I and the resulting fragments separated by pulsed field gel electrophoresis. Although the size of the fragments observed differs for the different bacteria, in each case 7 fragments are obtained. Suggest an explanation for these results.
You have isolated DNA of a 500 bp EcoRI restriction fragment and you would like to determine its restriction map. The enzyme HindIII cuts the fragment once to generate a 400 bp fragment (A) and a 100 bp fragment (B).
You label the 5' ends of the fragment with 32P, cleave with HindIII and isolate the labelled A and B fragments from a 5% polyacrylamide gel. You then generate partial digests of each fragment with HindII. Below is shown an autoradiogram of the resulting gel.
- (a) Draw the HindII map for the whole EcoRI fragment. Include the distances, in base pairs, between HindII, HindIII, and EcoRI sites.
Next, you decide to clone the EcoRI fragment into the EcoRI site of the M13 vector mp8. The structure of M13 mp8 is shown below.
- (b) How would you isolate clones which contain the EcoRI fragment? Describe briefly the steps required (i.e. enzymes needed, etc.)
- (c) Assume you were successful in isolating several independent clones. How would you determine the orientation of the fragment relative to the vector without doing any DNA sequencing?
A 1000 bp EcoRI restriction fragment contains a gene you are interested in. As a first step, you construct a restriction map of the fragment using the enzymes SmaI and HindIII. Below is shown an agarose gel of the appropriate digests.
Draw a restriction map of the fragment and show the distances, in base pairs, between the HindIII, SmaI, and EcoRI sites.
Next, you want to clone the three fragments from the HindIII + SmaI digest (labelled a, b, and c) into the vector pUC9 (shown below).
Starting with pure isolated DNA fragments and pUC9 DNA, describe how you would clone each fragment. Include in your answer:
- The restriction enzyme(s) you would use to cleave the vector.
- Other enzyme(s) you would need.
- How would you screen for potential clones on agar plates at the end of the experiment? Describe briefly the principle behind the screening procedure.
If you digested the Salmonella typhimurium chromosome with a restriction endonuclease that recognizes a 8 bp DNA sequence, how many DNA fragments would you expect to obtain? (The Salmonella typhimurium chromosome is about 4800 Kb.)
How often would you expect to find a 4 bp restriction site in a random DNA sequence?
You have purified a 1100 bp HindIII restriction fragment that you plan to sequence eventually. As a first step, you decide to construct a restriction map of the fragment for the enzymes EcoRI and SmaI. Below is shown an agarose gel of the appropriate digests.
- (a) Draw a restriction map of the fragment and show the distances, in base pairs, between the HindIII, EcoRI, and SmaI sites.
Next, you want to clone the three fragments from the EcoRI and SmaI double digest (labelled a, b, and c) into the M13 vector MP8 (shown on the next page). Starting with pure isolated a, b, and c fragments describe how you would clone each fragment.
- Indicate the restriction enzyme(s) you would use to cleave the vector for each cloning experiment.
- Indicate any other enzymes(s) you would need.
- Suppose that in your cloning of fragment a, you isolate several independent transformants and extract the single stranded DNA from phage isolated from them. How could you identify clones that contained opposite orientations of fragment a without sequencing the DNA or using restriction enzymes? (Some clones will contain the "Watson" strand in the phage DNA and some will contain the "Crick" strand. How can you tell them apart?)
Please send any comments, suggestions, or questions to firstname.lastname@example.org
Last modified October 31, 2003