Bacterial plasmids are closed double-stranded DNA molecules of approximately 200 kb in size. Plasmids generally contain genes specific for enzymes that are helpful in the bacteria’s survival within the host cell such as antibiotic resistance. A laboratory exercise involving the preparation of plasmid DNA from competent Escherichia coli (E. coli) was performed. Techniques in handling plasmids and digesting these with restriction enzymes were conducted. Quantification of plasmid DNA was performed using serial dilutions of the DNA solution collected. The approximate mass of DNA was computed based on the control fragments of pAMP. Single- and double-digestion of pAMP plasmids was performed in order to determine the banding patterns of each plasmid.
PLASMID MINIPREPARATION OF pAMP
Upon running a 0.8% agarose gel using three dilutions (1:10, 1:50 and 1:100) of HindIII-digested pAMP and HindIII-digested λ, staining the gel with ethidium bromide (1 ug/ul) and viewing the results using an ultraviolet transilluminator, we observed that the plasmids with the biggest dilutions showed very faint bands. On the other hand, the other dilutions of HindIII-digested pAMP and HindIII-digested λ showed brighter bands.
The lanes wherein the 1:50 dilutions of both plasmid digests were loaded showed brighter bands than that of the bands from the 1:100 dilutions. The bands located in the lanes wherein the 1:10 dilutions of HindIII-digest pAMP and HindIII-digested λ showed the brightest bands among the three dilutions. The bands generated from the pAMP digestion using HindIII had an approximate size of 4,539 bp while the bands generated from the λ digestion using HindIII had an approximate size of 4,361 bp.
In order to determine the mass of the λ DNA in the selected fragment, the size of the fragment was multiplied by the volume of DNA loaded onto the lane. The resulting product was then divided by the approximate size of the λ DNA. This can be described as follows:
Mass of λ DNAfrag = 4,361 bp X 15 μl
————————- = 16.35 ng/ul
When the mass of the selected λ DNA is multiplied by the dilution factor of the selected pAMP lane (0.01), the resulting value is 0.1635 ng/ul. This means that the lane with the faintest band in the pAMP samples contained approximately 0.1635 nanograms of plasmid DNA per microliter.
RESTRICTION ANALYSIS OF PURIFIED pAMP
Comparison of the two gel lanes containing miniprep DNA with the two lanes comtaining control pAMP showed almost the same banding patterns in terms of incubating the DNA samples in plain buffer with RNase and with the double digest composed of BamHI and HindIII. The same banding patterns suggests that the fragments resulting from the digestion of the miniprep DNA were of the same size as that of the control pAMP DNA that was digested with the same restriction enzymes.
However, there was a slight difference between the miniprep DNA fragment sizes from the control pAMP DNA fragment sizes wherein the uncut miniprep bands were slightly smaller than the uncut pAMP bands. This might be due to small differences in the total size of the plasmid DNA in the miniprep and the control pAMP (Duarte et al., 2007).
EDTA is used in the purification and restriction digestion of DNA because it is a chelating agent that binds free cations that are present in the DNA solution (Zhu et al., 2006). Unfortunately, the presence of EDTA is the Tris-EDTA buffer also inhibits that action of the restriction enzyme hence it is important to determine the optimal concentration of EDTA that can be used in laboratory experiments in order to both acquire the necessary chelating effect yet not hinder the digestion reaction of the restriction enzymes. EDTA is also helpful in inhibiting the action of DNases that might be present in the DNA solution. DNases are bacterial enzymes that degrade double-stranded DNA and these enzymes are released during the lysing of bacterial cells during the harvesting step of competent E. coli cells.
The map of a dimeric plasmid would be observed as follows:
The map of a dimeric pAMP plasmid with a head-to-tail recombination will be as follows:
The gel banding pattern resulting from the double digestion using BamHI and HindIII will be:
Duarte SP, Fortes AG, Prazeres DM and Marcos JC (2007): Preparation of plasmid DNA polyplexes from alkaline lysates by a two-step aqueous two-phase extraction process. J Chromatogr A. 1164(1-2):105-12.
Zhu K, Jin H, He Z, Zhu Q, Wang B (2006): A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis. Nat. Protoc. 1(6):3088-93.