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Advanced Alternatives Within Part II
Advanced Alternatives Within Part II ADVANCED ALTERNATIVES, PART II—SCREENING TRANSFORMANTS Depending on the changes made in Part I—Manipulation of DNA, the appropriate changes need to be made during this part of the lab project. The overall goal of the project is to confirm the cloning of the gene of interest into the pET-41a vector. In other words, do you have the correct coding sequence present and in frame to enable protein expression to occur in bacteria? In this part, we describe three ways to do this: PCR screening, restriction analysis of miniprep DNA, and DNA sequencing. Depending on whether egfp or a different gene was cloned in Part I, different primers and restriction enzymes might be needed for these experiments. In practice, DNA sequencing is the gold standard for this confirmation. To perform sequencing, plasmid DNA needs to be purified from individual clones following transformation. In the time it takes to purify the DNA, send samples for sequencing, and obtain the sequence data, other screening methods can be performed as well. While these other methods are not absolutely necessary, it is always useful to have multiple tools at your disposal. Unlike some of the Advanced Alternatives in Part I, downstream experiments in Part III are not affected by the options suggested here.
AA2.1.
Omit the PCR Screen
While screening by PCR can be particularly helpful in more difficult cloning procedures, the cloning of egfp described in this manual typically yields a significant percentage of positive clones. Students can immediately go from the experimental transformation plate and perform miniprep DNA purification, which can then be used for restriction analysis and DNA sequencing. Since a miniprep needs to be performed in order to have plasmid to sequence, doing a quick digest followed by DNA agarose gel analysis will indicate if the insert is present or not before sequencing.
AA2.2.
Omit the Restriction Analysis
Similar to “Omit the PCR Screen”, students may also leave out the restriction analysis. In this scenario, colonies from the experimental transformation plate can be used as templates for the PCR screen, then the same colony can be used for growing the overnight culture. It is important that students only touch each colony once, streaking the plate with the same tip used for adding the bacteria to the PCR reaction. This will ensure that the same clone is represented in both experiments. In general, the most important data is the sequencing data, which will confirm the sequence of the clone. However, with more challenging cloning projects, it can save time and money to perform the PCR screen prior to sequencing to verify those clones with the insert of interest.
AA2.3.
Choose Different Restriction Enzymes for Analysis
While the most common practice is to simply cut the insert from the plasmid using the same enzymes that were used for cloning, other enzymes could also be used to confirm the presence of insert. In this alternative, allow 43
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PART | II
Screening Transformants
students to select from a library of restriction enzymes and have them predict gel patterns for both plasmids with insert and plasmids without insert. The most effective method for this is choosing a single enzyme that gives a significantly different pattern in the presence of insert. Furthermore, there are many freely available online tools that analyze given sequences for restriction enzyme sites and generate predicted fragment numbers and sizes. Have students perform the agarose gel electrophoresis to determine if their results support the presence of the desired insert.
Additional materials Alternative restriction enzymes for students to choose from.
AA2.4.
Design Your Own Primers for the PCR Screen
For extra practice in the design of PCR primers, have students design primers for their PCR screen in a manner similar to the anchored screen in Lab Session 6c. General guidelines for PCR primer design are given in Lab Session 25.
Additional materials Custom oligonucleotide primers.
AA2.1.
Omit the PCR Screen
While screening by PCR can be particularly helpful in more difficult cloning procedures, the cloning of egfp described in this manual typically yields a significant percentage of positive clones. Students can immediately go from the experimental transformation plate and perform miniprep DNA purification, which can then be used for restriction analysis and DNA sequencing. Since a miniprep needs to be performed in order to have plasmid to sequence, doing a quick digest followed by DNA agarose gel analysis will indicate if the insert is present or not before sequencing.
AA2.2.
Omit the Restriction Analysis
Similar to “Omit the PCR Screen”, students may also leave out the restriction analysis. In this scenario, colonies from the experimental transformation plate can be used as templates for the PCR screen, then the same colony can be used for growing the overnight culture. It is important that students only touch each colony once, streaking the plate with the same tip used for adding the bacteria to the PCR reaction. This will ensure that the same clone is represented in both experiments. In general, the most important data is the sequencing data, which will confirm the sequence of the clone. However, with more challenging cloning projects, it can save time and money to perform the PCR screen prior to sequencing to verify those clones with the insert of interest.
AA2.3.
Choose Different Restriction Enzymes for Analysis
While the most common practice is to simply cut the insert from the plasmid using the same enzymes that were used for cloning, other enzymes could also be used to confirm the presence of insert. In this alternative, allow 43
44
PART | II
Screening Transformants
students to select from a library of restriction enzymes and have them predict gel patterns for both plasmids with insert and plasmids without insert. The most effective method for this is choosing a single enzyme that gives a significantly different pattern in the presence of insert. Furthermore, there are many freely available online tools that analyze given sequences for restriction enzyme sites and generate predicted fragment numbers and sizes. Have students perform the agarose gel electrophoresis to determine if their results support the presence of the desired insert.
Additional materials Alternative restriction enzymes for students to choose from.
AA2.4.
Design Your Own Primers for the PCR Screen
For extra practice in the design of PCR primers, have students design primers for their PCR screen in a manner similar to the anchored screen in Lab Session 6c. General guidelines for PCR primer design are given in Lab Session 25.
Additional materials Custom oligonucleotide primers.