Methods and reagents: Better competent cells and DNA polymerase contaminants Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a new method for harvesting Escherichia coli cells for transformation, and contaminants found in DNA polymerase stocks used for the polymerase chain reaction. For details on how to partake in the newsgroup, see the accompanying box. Over-the-hill E. coli ********************* Going, going, ...gone! A study concerning the time at which cells are harvested for transformation was discussed recently. An intriguing discovery was made that there are two peaks in the transformation competency of Escherichia coli cells during growth of the culture. During an attempt to improve the efficiency of transformation, the optical density at 600 nm (OD600) of E. coli cells was unexpectedly found to be optimal at two separate times during the growth cycle - at OD600 values of 0.4 and 0.94. The highest transformation frequencies were 4 x 10^7 transformants per microgram of pBR322 plasmid DNA at an OD600 of 0.4, and 7 x 10^7 at an OD600 of 0.94. Although variablity was seen when different host strains were used, they all showed a common feature: surprisingly, there is a valley between the two peaks, bottoming out at an OD600 of 0.7 to 0.8, indicating a loss of competency at this stage of the growth curve.[1] Netters are now trying their hand at catching the cells at the correct density for increased numbers of transformants, in the hope of squeezing out twice the number of transformants using this new information. Zophonias O. Jonsson (zjons@vetbio.unizh.ch) wrote that the main problem with this method, however, is the very small time window for collecting cells at the higher density. The competency of cells drops quickly above a density of 0.95, and collection of cells at OD600 values close to 1.0 using a Uvicon 810 spectrophotometer resulted in about one order of magnitude fewer transformants using the standard CaCl2 protocol [2] than if the cells had been collected at an OD600 of 0.4, as is practiced commonly and recommended from previous studies. [3] The optimal cell density can easily be missed if care is not taken when monitoring the growth of the culture, and it can be quite a challenge to catch the cells at an OD600 of 0.94-0.95 because the time between between measurements of 0.80 and 1.00 is only few minutes for the faster growing strains of E. coli, requiring some precision timing. Some others also thought it impractical to catch the cells at the later time, since readings taken on different machines can vary by as much as 5%. Unfortunately, the authors of the paper did not specify the type of spectrophotometer used in the study - a critical oversight. It is still a matter of speculation as to why there is such a dramatic change in the physiology of bacteria grown in culture that affects their ability to take in free DNA. It remains to be seen why loss of competency is observed while the cell density is between these two optima, other than the obvious conclusion of an onset of a bacterial mid-life crisis. The molecular reason for such an unexpected result may provide an insight into the artificially induced state of transformation competency. Contaminated Taq stocks *********************** When performing the polymerase chain reaction (PCR), some netters have reported seeing extra bands within their control samples without added DNA template when a sample of the PCR product is analysed by gel electrophoresis. It appears that different commercial Taq polymerase preparations have varying amounts of genomic DNA contamination. One netter wrote that the use of primers specific for bacterial ribosomal genes produced a discrete band during every attempt at PCR, and this band was particularly strong when a combination of high primer concentration (100 pmol of each) and a very low number of template DNA strands was used. The band did not appear to be a primer dimer and disappeared when increasing amounts of template were used. Another netter, who was amplifying a gene encoding a mitochondrial 12 S ribosomal RNA (rRNA), always observed a contaminating DNA band encoding a 16 S rRNA. Upon sequencing this, it became apparent that the gene was not from the organism studied, and for unknown reasons was 90% identical to the gene encoding Chromobacterium violaceum 16 S rRNA. When using a commercial source of PCR primers specific for screening a human yeast artificial chromosome (YAC) library that was constructed using a vector of pBR322 origin, Rob Preston (rapr@med.pitt.edu) found that the controls without any DNA added also showed a discrete band, and upon further investigation found the source of the contaminating band to be the polymerase stock itself. One likely possibility is that the enzyme had been extracted from an E. coli strain carrying a recombinant plasmid that was probably constructed by ligating the gene for DNA polymerase onto a derivative of this commonly used cloning vector. Sufficient plasmid DNA to cause amplification was then carried over into the concentrated polymerase stock during the purification process. Extraneous bands were also seen in another attempt to isolate PCR fragments using primers with a close resembance to vector sequences. To rid his reactions of the contaminating DNA, Dr Preston treated the PCR mixture lacking template and primers with RNase-free DNase I. An equimolar amount of EDTA was then added to chelate the Ca2+ and Mg2+, thus destabilizing the DNase I, which was then inactivated by heating the solution to 65 degrees C for 10 min before the cycling reactions. The Taq polymerase which, unlike DNase I, does not require free Mg2+ for heat stability, remained active under these conditions. This method was probably unnecessarily complicated because of the many steps involved and the need for further addition of MgCl2 for the PCR. Clean-up of the enzyme stock could have been done more simply, for example by UV inactivation of the contaminating DNA. Newly available ultra-clean Taq DNA polymerase preparations containing less genomic DNA should alleviate some of the unwanted extraneous bands in PCR. For example, Perkin-Elmer sells a polymerase (AmpliTaq[R] DNA polymerase, LD) specifically purified for amplification of low copy-number targets. This enzyme is touted as having no more than one copy of genomic DNA per reaction containing 2.5 units of enzyme. Furthermore, the use of specially designated `DNA-free' positive displacement micropipets and quality filtered tips may help reduce background problems that are due to laboratory vector contaminants. Obviously, care must be taken when interpreting PCR products using enzyme stocks from different companies, and the source of the enzyme must be considered when selecting primers. References [1] Tang, X. et al. (1994) Nucleic Acids Res. 22,2857-2858 [2] Inoue, H., Nojima, H., and Okayama, H. (1990) Gene 96,23-28 [3] Hanahan, D. (1983) J. Mol. Biol. 166,557-580 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/oct94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - Better competent cells and DNA polymerase contaminants. Trends in Biochemical Sciences 19(10):426-427. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************