Plasmid Identification
A plasmid is a small, circular, double-stranded DNA molecule, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. Plasmids are used as tools to clone transfer, and manipulate genes, these plasmids are called vectors. We can insert DNA fragments or genes into a plasmid vector, creating a recombinant plasmid. This plasmid can be introduced into a bacterium by way of the process called transformation (plasmid / plasmids). Then, because bacteria divide rapidly, they can be used as factories to copy DNA fragments in large quantities. When a bacterium divides, all of the plasmids contained within the cell are copied such that each daughter cell receives a copy of each plasmid.
To use a plasmid as a factory to copy DNA, we need to recognize the code of the gene (section of the plasmid to copy) that we want to copy, but how to do it? Well, here is where some guys, called Restriction Enzymes, take place. A Restriction Enzyme cuts DNA at specific recognition nucleotide sequences known as restriction sites, these restriction sites are the codes of the genes that we want to copy (Restriction enzyme).
In order to determine if I have the best plasmid to duplicate a gene, I can use a Gel Electrophoresis: they are used to separate macromolecules like DNA, RNA and proteins. DNA fragments are separated according to their size. Proteins can be separated according to their size and their charge. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge (What is gel electrophoresis?). When the current starts to flow, the fragments are being carried across the gel. Given the composition (concentration of agarose) of the gel, the smaller fragments migrate further and the bigger ones doesn’t go too far from the loading cell.
In this experiment I’m going to test my ability to recognize a unknow plasmid with the code 3377698, by using past lab protocols: I’m going to use the knowledge earned through the year and the protocols of the last labs to perform the identification of a DNA sample (pKAN, a pAMP, or a pBLU), which is supplied in TE buffer (10mM Tris-HCI, pH 8.0, 1mM EDTA), with a single and a double digestions. I’ll use 3 types of enzymes (Pstl-HF ,Hindlll-HF and BamHI-HF) two of them for a double digestion, and the other one for a single digestion: a single digestion won’t be enough to tell the type of plasmid that I’m trying to identity, a single digestion along with a double digestion will give enough data to determine the kind of plasmid. To achieve this goal I have to determine the DNA concentration of my sample using a Bio-Rad Spectrophotometer, make a single restriction digestion and a double restriction digestion, prepare and use 1X TAE Buffer to make a 0.7% agarose gel, analyze the digestions using Gel Electrophoresis, and determine the fragment sizes by semi-log plot.
Methods
The method section will be separated into 3 parts
Part 1
In order to start with the identification of the plasmid 3377698, I needed to know the concentration of DNA in our sample. To measure the amount of DNA in the sample, I used a Bio-Rad Spectrophotometer.
Materials
- trUView cuvettes
- pGLO Plasmid DNA
- Elution Buffer
- dH2O
Procedure
- The spectrophotometer was setted up for DNA/RNA assay, and the type of nucleic acid was dsDNA. The conversion factor for the sample was A260 1.0=50 μg/mL. A blank cuvette was prepared with the next specifications:
57 μL of dH20 and 3μL of the elution buffer were added and mixed to a clean cuvette, by gently pipetting the mixture up and down.
The cuvette was loaded onto the machine in the correct orientation.
2.After to Read the Blank, the samples were prepared, and read.
A 1/20 dilution sample was prepared by adding 3 μL of DNA sample to 57 μL of dH2O. Mixed well by pipetting up and down, but avoiding the generation of any air bubbles in the sample.
The cuvette was loaded onto the machine in the correct orientation.
4. The “Conc” value was recorded, and multiplied by the dilution factor (20) to get the concentration of the sample to be identified.
Note: the concentration was given in ug/mL, which is the same as ng/μL “Conc” value: 4.7864 ng/μL.
To get the purity of the DNA sample, the “A260/280” assay option was selected.
Notes: A radio between 1.8–2.0 denotes very clean DNA; lower than 1.8 indicates the presence of proteins and/or other contaminants. A radio higher than 2.0 indicates that the samples may be contaminated with chloroform, phenol, or have high amount of RNA.
Part 2
For this experiment, two types of restriction digestions were used; a single restriction digest and a double restriction digest (the difference between a single restriction digest and a double restriction digestion is that for the single restriction digest a single enzyme is used, and for a double restriction digestion, two enzymes are used).
Materials
- pGLO Plasmid DNA
- Pstl- HF
- Hindlll-HF
- BamHI-HF
- 10X CutSmart Buffer
- Gel Loading Dye Purple 6x
- dH2O
- PCR tube
Procedure
- The needed volume of DNA solution were calculated to make it equal to 1,000 ng (1ug) of DNA solution. The calculations were based on the concentration of the extracted DNA from parts 1 of this experiment.
95.728/1μL = 1,000 ng/x
= 10.44626442 μL
2. The value for x found it in the previous step was the volume of DNA solution that was needed to be added to the mixture for the single digestion and the double digestion.
3. The data tables for each one of the digestions are in the section below. Each digestion had their own volumes of: enzymes, dH2O, 10X CutSmart Buffer and DNA solution (x value), but the procedure was the same. The reagents in the table 1.1 were added together into a PCR Tube in the order they occur for a single digestion, and the reagents in the table 1.2 were added together into another PCR Tube for a double digestion (the recipes for this digestions were supplied by Nebcloner — http://nebcloner.neb.com).
4. After to put the reagents into the PCR Tubes, the PCR Tubes were gently flicked, then they were spun briefly in a microfuge to collect any solution that could have been on the walls of the tubes.
5. The samples were placed in the PCR machine. The PCR machine was programed to incubate the reaction at 37 C for 1 hour, then heat-inactivate the enzymes by incubating at 80 C for 20 minutes, then finally hold at 4 C (for infinite)
6. After the reactions were completed, 1 μL of Gel Loading Dye Purple 6x were added to each one of the digestions, and the samples were stored at -20 C. Also the DNA solution was stored at the same temperature for future references.
Part 3
In order to analyze the DNA and determine the kind of plasmid that it was, a Gel Electrophoresis was needed.
Materials
- Digested DNA (with loading dye added)
- 1000 bp Marker Ladder
- 0.7% Agarose Gel with GelRed Stain
- Gel Electrophoresis Box
Procedure
- 0.35 g of agarose were melted in 50 mL 1X TAE Buffer to create a 0.7% agarose solution (The 1X TAE Buffer was prepared by using the formula C1V1=C2V2: 1000 mL of 1X TAE Buffer solution were prepared with 20 mL from a 50X TAE Buffer stock solution, by pouring those 20 mL of 50X TAE Buffer stock solution into a graduated cylinder, and then adding dH2O to bring the final volume to 1000 mL).
- The agarose solution was allowed to cool in the flask for about 5 minutes.
- 5 μL GelRed Stain were added to the flask of melted agarose. The mixture was swirled in the stian.
- The agarose solution was poured into the prepared gel tray and allowed to cool at least 20 minutes before be used.
- The flak was immediately cleaned with hot water after pouring the gel.
- 10 μL of the single, and double digestions DNA were loaded in the wells, -but separated-. This included the loading dye already added (1.5 μL of loading dye and 8.5 μL of the DNA sample).
- 8 μL of the 1000 bp Marker Ladder were loaded along with the digestions.
- The gel ran for an hour at 100 v, then the gel was photographed under ultraviolet light.
Results
For this experiment the expected size, base pairs (bp) of the bands were different on each type of plasmid: depending on the kind of plasmid the enzymes were going show different band sizes on each digestion:
The figure 1.1 shows the expected band sizes for a single restriction digestion with the same enzyme on the three possible plasmids.
The figure 1.2 shows the expected band sizes for a double restriction digestion with the same enzymes on the three possible plasmids.
The gel with the sizes of the bands for the 1000 bps Ladder, single digestion, and double digestion are shown on figure 1.3.
The sizes of the bands for the double and single digestion on figure 1.3, were supported by the semi log-plot graph 1.1


The data on graph 1.1 comes from the table 1.3: where the sizes of the bands for the Marker Ladder, and the bands of the restriction digestions are found. The Marker Ladder was graphed first, then the values of the sizes for the bands of the restriction digestions were calculated from the equation of the best-fit Line, and using the distance traveled by each band as the value for X.

Conclusion
The results of the experiment were successful: the data obtained from the gel was positive for a pAMP plasmid. The expected bands for a pAMP plasmid that was single digested with the enzyme Pstl-HF were only one, this band should be at 4539 bp according to the figure 1.1 on the results section. In this experiment the band obtained from the single restriction digestion with the enzyme Pstl-HF was 4513 bp, which was the first indicator of a pAMP plasmid, but given that a single digestion would not be enough to ensure anything; I had to include the results of the double restriction digestion: the expected bands for a double digestion with the enzymes Hindlll-HF and BamHI-HF were 3755 bp and 784 bp respectively. The bands for the double digestion on this experiment were 3572 bp for the band of the enzyme Hindlll-HF and 750 bp for the band of the enzyme BamHI-HF. Therefore I can assume that the DNA sample with the code 3377698 was a pAMP plasmid.
There is no way to accurately know the real size of the bands on the gel. In order to calculate the size of a band, I had to measure the distance traveled by each band with a millimeter ruler. I used the middle of the loading well as my reference point to measure the distance traveled by the bands, also I used the middle of the bands as the final point for the distance traveled by the bands. This technique didn’t allowed me to get a better data to calculate the sizes of the bands: the lack of accurate tools was a reasonable factor that didn’t allowed the correct calculation of the bands sizes.
Although my protocol: recipe, and concentration of the DNA sample, were correct. I had to deal with some problems:
- Possible contamination of used enzymes: the used enzyme for the single digestion was Pstl-HF, and I assumed that there was a polluting agent on the supplied enzyme, because on the figure 1.3 of the results section, the band for the single digestion showed an unusual band, like if the sample was contaminated with chloroform, phenol, or an other enzyme. My suspicion is that given the supplied enzyme was used for multiple people, someone accidentally could had mix some contaminant with the enzyme. I gave credit to this hypothesis because more people that used the same enzyme had the same problem.
- Misuse of the enzymes: initially the experiment was designed to perform a double digestion with another enzyme (Sacl-HF), but this enzyme wasn’t present/ didn’t cut on this kind of plasmid. I didn’t realized that this enzyme didn’t cut all of the possible kinds of plasmids, so I performed the experiment with this enzyme and may bands on the gel were unclear and unexpected. As a solution I decided to use a different recipe with different enzymes for my double digestion. Another possibility for this problem was the possible use of the wrong enzymes; my teammate told me that he had the same problem with the same enzymes, and he assumed that the real problem was that the supplied enzymes were different from what they were actually marked. I decided to adjust my experiment to use different enzymes and all the changes were added to the final report.
Note: for future experiments always double check my protocol (recipe, enzymes, concentration, buffer, etc.) before to start with the experiment.
References
(plasmid / plasmids) https://www.nature.com/scitable/definition/plasmid-plasmids-28
(Restriction enzyme) https://en.wikipedia.org/wiki/Restriction_enzyme
(What is gel electrophoresis?) http://www.yourgenome.org/facts/what-is-gel-electrophoresis
The recipes for the restriction digestions were supplied by NEB Cloner (http://nebcloner.neb.com/#!/)
The expected sizes of the bands on the gel were supplied by NEB cutter (http://nc2.neb.com/NEBcutter2/)
The DNA sequences were supplied by DNALC (http;//www.dnalc.org/resources/plasmids.html)