Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

General Laboratory Procedures, Equipment Use, and Safety Considerations 

I. Safety Procedures

II. Preparation of Solutions

III. Disposal of Buffers and Chemicals

IV. Equipment

V. Working with DNA

A. Storage .

The following properties of reagents and conditions are important considerations in processing and storing DNA and RNA. Heavy metals promote phosphodiester breakage. EDTA is an excellent heavy metal chelator. Free radicals are formed from chemical breakdown and radiation and they cause phosphodiester breakage. UV light at 260 nm causes a variety of lesions, including thymine dimers and cross-link. Biological activity is rapidly lost. 320 nm irradiation can also cause cross-link, but less efficiently. Ethidium bromide causes photo oxidation of DNA with visible light and molecular oxygen. Oxidation products can cause phosphodiester breakage. If no heavy metal are present, ethanol does not damage DNA. Nucleases are found on human skin; therefore, avoid direct or indirect contact between nucleic acids and fingers. Most DNases are not very stable; however, many RNases are very stable and can adsorb to glass or plastic and remain active. 5 E C is one of the best and simplest conditions for storing DNA. -20 deg C: this temperature causes extensive single and double strand breaks. -70 E C is probable excellent for long-term storage. For long-term storage of DNA, it is best to store in high salt ( >1M) in the presence of high EDTA ( >10mM) at pH 8.5. Storage of DNA in buoyant CsCl with ethidium bromide in the dark at 5 E C is excellent. There is about one phosphodiester break per 200 kb of DNA per year. Storage of λ DNA in the phage is better than storing the pure DNA. [ ref: Davis, R.W., D. Botstein and J.R. Roth, A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. 1980.]

B. Purification .

To remove protein from nucleic acid solutions:

1. Treat with proteolytic enzyme, e.g., pronase, proteinase K
2. Purify on a silica-based column such as a Qiagen PCR Prep Column
3. CsCl/ethidium bromide density gradient
4. Phenol Extract. The simplest method for purifying DNA is to extract with phenol or phenol:chloroform and then chloroform. The phenol denatures proteins and the final extraction with chloroform removes traces of phenol
5. Purify on silica-based column such as Qiagen Brand columns (http://www.qiagen.com)

C. Quantitation .

1. Spectrophotometric. For pure solutions of DNA, the simplest method of quantitation is reading the absorbance at 260 nm where an OD of 1 in a 1 cm path length = 50 μ g/ml for double-stranded DNA, 40 μ g/ml for single-stranded DNA and RNA and 20-33 μ g/ml for oligonucleotides. An absorbance ratio of 260 nm and 280 nm gives an estimate of the purity of the solution. Pure DNA and RNA solutions have OD 260/OD 280 values of 1.8 and 2.0, respectively. This method is not useful for small quantities of DNA or RNA (<1 μ g/ml).
2. Ethidium bromide fluorescence. The amount of DNA is a solution is proportional to the fluorescence emitted by ethidium bromide in that solution. Dilutions of an unknown DNA in the presence of 2 μ g/ml ethidium bromide are compared to dilutions of a known amount of a standard DNA solutions spotted on an agarose gel or Saran Wrap or electrophoresed in an agarose gel.

D. Concentration .

Precipitation with ethanol. DNA and RNA solutions are concentrated with ethanol as follows: The volume of DNA is measured and the monovalent cation concentration is adjusted. The final concentration should be 2-2.5M for ammonium acetate, 0.3M for sodium acetate, 0.2M for sodium chloride and 0.8M for lithium chloride. The ion used often depends on the volume of DNA and on the subsequent manipulations; for example, sodium acetate inhibits Klenow, ammonium ions inhibit T4 polynucleotide kinase, and chloride ions inhibit RNA-dependent DNA polymerases. The addition of MgCl 2 to a final concentration of 10mM assists in the precipitation of small DNA fragments and oligonucleotides. Following addition of the monovalent cations, 2-2.5 volumes of ethanol are added, mixed well, and stored on ice or at -20 E C for 20 min to 1 hour. The DNA is recovered by centrifugation in a microfuge for 10 min (room temperature is okay). The supernatant is carefully decanted making certain that the DNA pellet, if visible, is not discarded (often the pellet is not visible until it is dry). To remove salts, the pellet is washed with 0.5-1.0 ml of 70% ethanol, spun again, the supernatant decanted, and the pellet dried. Ammonium acetate is very soluble in ethanol and is effectively removed by a 70% wash. Sodium acetate and sodium chloride are less effectively removed. For fast drying, the pellet can spun briefly in a Speedvac, although the method is not recommended for many DNA preparations as DNA that has been over dried is difficult to resuspend and also tends to denature small fragments of DNA. Isopropanol is also used to precipitate DNA but it tends to coprecipitate salts and is harder to evaporate since it is less volatile. However, less isopropanol is required than ethanol to precipitate DNA and it is sometimes used when volumes must be kept to a minimum, e.g., in large scale plasmid preps.

E. Restriction Enzymes

Restriction and DNA modifying enzymes are stored at -20 deg C in a non-frost free freezer, typically in 50% glycerol. The enzymes are stored in an insulated cooler which will keep the enzymes at -20 deg C for some period of time. The tubes should never be allowed to reach room temperature and gloves should be worn when handling as fingers contain nucleases. Always use a new, sterile pipet tip every time you use a restriction enzyme. Also, the volume of the enzyme should be less than 1/10 of the final volume of the reaction mixture.

 VI. Sterile Technique

1. All media, including plates, liquid media and top agar must be autoclaved immediately after it is prepared. It is best to prepare media in several small bottles, only opening one at a time. Check the bottle for contamination before you use it by gently swirling it and looking for cloudy material in the center. Always grow up a small amount of broth alone when growing cells overnight. A small amount of contamination is not always evident until the media is incubated at 37 deg C.
2. Use a flame on inoculating loops and on the lips of media bottles before and after pipetting from them. Never leave a media or agar bottle open on the bench and don's take an individually-wrapped pipet out of its protective wrapper until you are ready to use it (i.e., don't walk across the room with an unwrapped pipet). Always use a fresh, sterile pipet or pipet tip when pipetting culture media, and never go back into a media bottle or cell culture with a used pipet.
3. To prevent wide-scale, untraceable contamination, each person should have his own stock of liquid culture media, top agar, plates, 100% glycerol, glycerol stocks of cells, etc. and don't share.
4. Overnight cultures should be grown only from a single colony on a fresh plate or from a previously-tested glycerol stock that was grown from a single colony. To prepare an overnight culture from a glycerol stock, take an individually-wrapped 1-ml pipet and a culture tube of media to the -80 deg C freezer. Quickly remove the cap from the freezer vial containing the glycerol stock, scrap a small amount of ice from the surface of the culture, replace the cap on the freezer vial, and place the pipet into the culture tube. Sufficient numbers of bacteria are present in the ice in order for the culture to grow to saturation in 16 hours. Never let the glycerol stock thaw.
5. Think about what you are doing. The best defense is common sense.

VII. Working with E. coli

A. Small Scale Cultures

Experiments using E. coli cells should always be done on fresh cultures, either from a freshly streaked plate or from a glycerol stock. To grow a small scale E. coli culture, prepare 3-5 ml of LB (or appropriate broth - include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. (Note: smaller tubes can be used but the culture will not be appropriately aerated and hence will not grow well and is not recommended). Inoculate one tube with a single colony from a fresh plate or a scraping from a glycerol stock. The second tube is used as a broth control. Incubate both tubes at 37 deg C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Do not allow cells to overgrow. Use immediately. For some applications, cells can be stored at 4 deg C for short periods prior to use.

B. Permanent Storage

For every culture used, in particular, for newly constructed strains or for cells containing plasmids, a permanent glycerol stock must be prepared as soon as the construct has been confirmed and this stock must be placed in the laboratory stock collection with the appropriate documentation and location information. Failure to follow these procedures will result in serious penalties. This procedure pertains not only to E. coli but to any organism for which a deep freeze stock can be prepared. Also, all plasmid constructs, including construction intermediates, must be maintained in cells, not as naked DNA stocks. For each construct, at least 2 stocks should be made. To prepare a glycerol stock for E. coli cells, combine 1.4 ml of a freshly grown overnight culture with 0.6 ml of sterile 50% glycerol. Mix well. Transfer to two freezer vials labeled with the strain name, the date and your initials (not an eppendorf tube). Immediately place into a dry ice/ethanol bath or into a box in the -80 deg C freezer. Note the location and enter data into the strain book.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010