Portland Community College | Portland, Oregon

An important and routine activity in DNA work involves removing a segment of DNA from one place, usually a plasmid or bacteriophage, and inserting into a different stretch of DNA  usually a plasmid or modified virus. One then isolates and characterizes the “cloned” product. This operation (subcloning to create new combinations of DNA sequences) is critical for basic research swell as research and development of new produces and procedures.  While the actual ligation and transformation steps can be carried out in a single afternoon, the planning and preparation of insert fragment and vector, and , at the other end of the procedures, the analysis of the results, are considerably more involved. They also require knowledge of  with recombinant principles and strategies, and skill at performing a variety of  calculations and analytical procures. These are embedded in, and necessary component of the single outcome for this course.

Upon completion of the course students should be able to:

• Construct and analyze a new plasmid prepared by the subcloning of a restriction fragment from one plasmid to another
• Review DNA sequences and select the correct restriction enzyme for matching recognition sites
• Produce new DNA constructs through proper use of DNA ligase
• Analyze plasmid properties and determine the correct plasmid choice for goals of protein expression or replication
• Follow a protocol to successfully select transformed bacteria 
• Use electrophoresis to analyze DNA fragment sizes and to determine orientation of inserted DNA within plasmids 

Students will work through two complete subcloning operations.  They will prepare, purify and quantitate both insert and vector, and following ligation and transformation, identify and characterize transformants.  The supporting lecture will provide an opportunity to discuss the theory, strategies and variations which apply to each part of the procedure. The second round of cloning will employ different vectors and/or fragments, and involve more student initiative in planning and execution

Produce bacterial clones containing the desired inserted fragments, along with the data necessary to show that the insertion was successful and in the desired direction (or not). (Note:  an unsuccessful ligation will suffice if the procedures analysis have been carried out, documented and interpreted correctly

Maintain a laboratory notebook which documents all procedure carried out in the lab that can be read and understood by a reader “skilled in the art” and which includes all necessary details of the procedures and analyses.

Participate in, and contribute to, all class and team discussions and activities;  Write all schedule examinations

Themes – Topics that thread through all of the instruction

• Care for procedure
• Project planning
• Alternative strategies

Concepts --  Things that need to be known and understood in support of the outcomes

• Purification of DNA
• Quantification of DNA
• Ligation
• Transformation
• Cloning
• Restriction analysis
• Southern blot analysis
• PCR analysis

Issues – Things that may need to be “overcome” in order to achieve the outcomes

• Maintaining laboratory notebook for continuity and detail
• Communication
• Controls
• Dealing with unexpected results

Skills –  Mastered by repletion and practice in order to achieve the outcomes 

1.     Determine the appropriate volumes of DNA, water, buffer(s) and enzyme for digesting the  necessary amount of  plasmid; explain the rationale behind the specific conditions chosen for the enzyme digest

2.     Calculate the amount of any particular restriction fragment to be generated from the digest  of a given amount of plasmid on which the restriction sites are known.

3.     Determine with reasonable accuracy the size of a DNA fragment using agarose gel electrophoresis

4.     Describe and carry out alternative methods for purifying fragments of DNA from common “Contaminants”(other DNA fragments, proteins and enzymes, RNA, agarose and salts)

5.     Determine the approximate concentration of fragmented plasmid DNA using gel electrophoresis with ethidium bromide stating and typical DNA standards

6.     Explain the use of alkaline phosphatase in the preparation of cloning vectors.

7.     Describe several strategies for creating compatible ends.

8.     Explain the difference between and consequences of directional vs. non-directional cloning.

9.     Setup a ligation reaction, including appropriate controls.  Explain the rational for selecting particular parameters for the reaction (DNA concentration, vector:insert ratio, amount of enzyme, buffer components, time and temperature of the reaction, and controls).

10.  Transform competent bacteria with ligated plasmid, including appropriate controls.

11.  Plate transformed bacteria.  Determine transformation efficiency.

12.  Pick colonies to prepare “mini-preps” of plasmid DNA for analysis.

13.  Plan and carry out restriction digests that twill allow analysis of transformation results (identify clones containing insert and determine the orientation of the insert with respect to the vector sequence).

14.  Carry out capillary transfer of DNA to nitrocellulose or nylon membranes, and demonstrate understanding of the principles and practices involved. Describe at least two methods for labeling DNA, and also for detection of labeled probes. Carry out hybridization, wash and detection of DNA probe on Southern Blot.

15.  Setup, carry out and interpret the results of a PCR reaction designed to identify clones containing insert, and determine the orientation of insert with respect to the vector sequence.

16.  Purify plasmid from cultured bacteria in large scale using ultracentrifugation in CsCl.

Interpret, research and describe a technical innovation in cloning strategy promoted by one of the molecular biology material suppliers.