Lab Topics for Fall 2005:

WEEK DATE               TOPIC                

1.         Aug 23-24         Introduction, pipetting, DNA structure and strand arrangement, prepare media and LB plates

The LB broth and agar plates are made according to the recipe in your protocol list.  This is a standard media for the growth of E. coli in molecular biology applications and is sterilized by autoclaving prior to use.  The agar is usually made selective for antibiotic-resistant bacteria by adding one or more antibiotics, such as ampicillin (which we will utilize), prior to pouring the molten agar into the plates.  However, the agar must be cooled before the ampicillin is added.  This means there will be a window of time between which the agar is too hot for ampicillin, and too cool to pour, so we have to monitor the agar to be sure it is at the right temperature for our application.

2.         Aug 30-31         Mini-prep DNA isolation, restriction enzyme digestion                                        

“Mini-prep” is a term commonly applied to the isolation of plasmid DNA from bacteria.  Plasmids are extrachromosomal genetic elements that are exploited for recombinant DNA cloning, so that the bacteria can produce large amounts of DNA fragments that can be manipulated and isolated in the lab.  The technique is based on the following series of events:  (1) disruption of bacterial cell wall/membranes; (2) denaturation of proteins and chromosomal DNA; (3) removal of denatured products; (4) selective precipation of small, plasmid DNA in alcohol.

Restriction enzyme digestion will be discussed at length in lab and lecture, but is essentially the use of microbial enzymes to cleave doublestranded DNA into fragments that are sufficiently small to be analyzed by gel electrophoresis.  Because the cleavage sites are sequence-specific, they can be both predictable and diagnostic.  Furthermore, the cleaved fragments can be rejoined by the DNA ligase enzyme, creating a system for cutting and pasting DNA fragments into various combinations (the basis for Recombinant DNA Technology).

3.         Sep 6-7             Gel electrophoresis, restriction mapping of unknown DNA

Gel electrophoresis is the process of using electric forces to drive a charged molecule (in this case, the DNA) through a matrix.  The matrix might consist of agarose gel or other materials such as polyacrylamide.  In this lab, we will utilize agarose gels because they provide resolution over a broad range of DNA fragment lengths.  During electrophoresis, the DNA is size-separated because small (shorter) fragments can move through the porous material more rapidly than larger (longer) fragments.  There is an additional dimension to consider with plasmid DNA, because in its native state it is circularized.  The circular nature of plasmids provides three possible conformations to occur during electrophoresis, each with different migration patterns:  (1) closed, circular DNA which can be supercoiled; (2) nicked plasmid DNA, which is still circular but is “relaxed” because the phosphodiester backbone on one strand has been broken, thus supercoiling cannot occur; (3) linear plasmid DNA, which has a double-strand break and migrates proportionally to its size.

4.         Sep 13-14         Fish DNA amplification, Exam 1 review 

This is the first time we will utilize the Polymerase Chain Reaction.  We will discuss this powerful, ubiquitously-utilized technique at length during class time.  The basis of this lab will be to use multiple primer sets to develop an appreciation for the specificity of the reaction, and the importance of primer choice during experimental set up. 

5.         Sep 20-21         Exam 1 given during lab meeting        

6.         Sep 27-28         Gel electrophoresis and ligations of PCR clones, transform competent DH5alpha cells

The ligation of our PCR clones to a plasmid exploits the peculiar observation that Taq polymerase adds an adenosine residue to the 3’ end of PCR products, independent of the template sequence.  Since each product has 3’ A overhangs, a technique has been developed to ligate these products to a vector that contains 3’ T overhangs.  The weak complementarity between the T and A overhangs is sufficient to expedite ligation of the product to the vector, and is commonly referred to as “T/A Cloning.” 

Once a recombinant plasmid has been made, we will induce E. coli cells to take up the plasmid and copy it as they reproduce.  The process of introducing a plasmid to bacteria is called “Transformation.”  Not all bacterial cells are capable of taking up DNA, so those which have been chemically prepared to do so are termed “competent” cells, and although the process is not well understood it involves permeability of the membrane and seems limited (in most cases) to one plasmid per cell.  The cells we will use are a well-characterized strain of E. coli called DH5a cells, because they allow alpha-complementation which refers to the mechanism of Blue/White screening.  Blue/White screening will be discussed in lecture and lab.

7.         Oct 4-5             Mini-preps, set up restriction digests

8.         Oct 11-12          Gel electrophoresis and analysis, cell cycle/general review for Exam 2                                        

9.         Oct 18-19          Exam 2 given during lab meeting                                                        

10.        Oct 25-26          Roadkill            DNA preparation, PCR amplification of actin sequences

We will be utilizing a new technique this year to characterize DNA from dead animal specimens.  The technique involves breaking down the tissue matrix using liquid nitrogen pulverization, then boiling the cells to release their contents (including DNA).  We will not truly isolate the DNA as a pure chemical in this process, instead we will add a reagent called Chelex that will chelate inhibitory factors out of our mixed solution.  Once these factors are eliminated, a very dilute concentration of DNA can be subjected to PCR amplification using primers for the actin gene family.  We are interested in the evolutionary differences between actin family members across taxa such as reptiles, amphibians, birds, mammals, and fish.

11.        Nov 1-2             Bioinformatics exercise

Bioinformatics is the field of science that collaboratively utilizes computer science, biology, genomics, and sequence analysis to derive information on evolution, disease, basic biological processes, etc.  The field revolves around the ever-expanding database of genomic information that is publicly availably through federally and privately-funded operations on the internet.  This exercise will be done sans professor, through a self-guided tour of the many resources available to you.  I will be giving you a detailed worksheet during this lab to complete and return by the end of the day.

12.        Nov 8-9             Electrophoresis of mammal PCR samples, distance calculation, Exam 3 review                                      

13.        Nov 15-16         Exam 3 given during lab meeting                    

14.        Nov 22-23         Thanksgiving Break    

15.        Nov 29-30         Human DNA isolation, TPA allele analysis for heterozygosity/homozygosity

We will use the chelex isolation again, this time without liquid nitrogen, to rapidly acquire human DNA from your cells.  The DNA will be amplified using primers that recognize a known polymorphism in the Tissue Plasminogen Activator gene (TPA).  The polymorphism, an Alu element insertion, is present in a significant portion of the population and many individuals will be heterozygous for the polymorphism.  We will analyze the results during our last meeting.

16.        Dec 6-7             Electrophoresis of TPA samples, Hardy-Weinberg exercise, Final Exam review