ABSTRACT:
A plasmid DNA is an extra-small circular structure commonly found in bacteria which is capable of replicating by itself in the cell. These are used in genetic engineering as vehicles to carry and make copies of a particular gene which can produce desirable effect. In this report, we discuss the isolation and purification of DNA fromEGFP-LC3 plasmidusing a modified alkaline lysis method and QIAprep spin columns. The concentration of the isolated EGFP-LC3 plasmid DNA was found to be 82.868µg/ml. The ratio of ODs taken at A260nm/A280nm was found to be 5.314 which could be because improper washing step which has resulted in the contamination of the RNA with degraded RNA in the sample. Restriction digestion was performed on the plasmid DNA using EcoRI restriction enzyme and the product was analyzed using agarose gel electrophoresis. The size of the plasmid DNA was found to be 4.5kbp.
INTRODUCTION:
Plasmids are small circular double stranded DNA molecules which are functionally independent from the main chromosomal DNA in a bacterial cell.They vary in size from 1 kb to 1000 kb in length. During cell division, the plasmid DNA is transferred along with the main chromosome into successive generations. The plasmid DNA is comprised of three components namely the origin of replication, an antibiotic resistance gene and a cloning site which is used to clone the gene of interest for genetic engineering [1-2]. Plasmids are commonly used as vehicles in order to genetically modifya cell by introducing the gene of interest to produce desirable proteins or to create resistance to a particular disease [2-3].
In this report, we discuss the isolation and purification of EGFP-LC3 plasmid DNA from an E.coli culture using modified alkalinelysis method and QIAprep spin columns. The alkaline lysis buffer contains a base (sodium hydroxide (NaOH)) and a detergent (sodium dodecyl sulphate (SDS)) which lyse the cell and releasesthe DNA into the solution. The DNA lysate when applied to the QIAprep spin columns, under high salt concentration binds to the silica membrane in the spin columns. The impurities are washed through the membrane into the collection tube using a buffer containing alcohol and the clean DNA is eluted using buffer EB (10 mM Tris-Cl, pH 8.5) [4-5].
The DNA is capable of absorbing UV light efficiently which allows it to be detected and quantified by a UV spectrophotometer. The absorbance is usually measured by taking the reading at A260nm (for DNA) and A280 nm (for protein). In order to determine the purity of the DNA, the ratio between the 2 absorbance values is usually calculated. The DNA concentration was calculated using Beer Lambert’s law. Beer Lamberts law is defined as the linear relationship among the absorbance and concentration of the nucleic acids electromagnetic radiations. The formula for Beer Lamberts law is A= εlc, wherein A indicates absorbance, ε indicates the molar extinction coefficient, l is length of the path light which has to move in the nucleic acid solution in centimeters and c indicates the concentration of the solution containing the nucleic acid [6].
In order to determine the size of the plasmid DNA restriction enzyme digestion was performed using the EcoRI restriction enzyme. Restriction enzymes are capable of recognizing specific sequence which are palindromic present in the DNA and cleave the DNA (phosphodiester bond) to produce fragments which are commonly called as restriction fragments. For the estimation of the size of the DNA fragments, agarose gel electrophoresis was performed. This is a common techniques used in the molecular laboratory for the separation of charged molecules like the DNA, RNA and proteins with different sizes. The agarose gel electrophoresis used an electric field where the negatively charged DNA moves from anode (negative) to cathode (positive) end.
FLOWCHART OF PROTOCOL
DNA was extracted from plasmid DNA using modified alkaline lysis method and QIAprep spin columns
Purity of the extracted DNA was estimated using UV spectrophotometer by measuring the absorbance at A260nm (for protein) and A280nm (for DNA)
DNA concentration was calculated using Beer Lambert’s law (A= εlc)
Restriction digestion was performed using ECORI enzyme
Agarose gel electrophoresis was performed to analyze the restriction digestion product and to determine the size of the linear plasmid DNA
MATERIALS AND METHODS:
For EGFP-LC3 plasmid DNA isolation [4-5]:
Materials
- E.coli culture
- EGFP-LC3 plasmid (AddGene) with a CMV promoter and an Ampicillin resistant gene.
- QIAprep® Miniprep kit comprising of the following-
- Resuspension buffer (Buffer P1) containing 50mM Tris-HCl,10mMEDTA and 100µg/ml RNase A.
- Lysis buffer (Buffer P2) containing 200 mMNaOH and 1% SDS.
- Neutralization Buffer (Buffer N3) containing 4M Guanidine hydrochloride and 0.5M Potassium acetate with pH 4.2.
- Wash buffer (Buffer PE) containing 20mM NaCl, 2mMTris-HCl and 80% ethanol with pH 7.5.
- Binding buffer (Buffer PB)containing 5M guanidine hydrochloride, 20mM Tris-HCl and 38% ethanol with pH 6.6.
- Elution buffer (Buffer EB) containing10mMTris-HCl with pH 8.5.
- 100% ethanol (Molecular grade)
- Microcentrifuge tubes (1.5ml capacity)
- Sterile pipette tips
- Microcentrifuge (Speed: 13,000 rpm)
- Vortex
- Appropriate personal protector (e.g. lab coat, disposable gloves, goggles, etc)
Methods:
- About 1.5ml of the overnight culture of E.coli in Liquid Broth medium was transferred into two separate microcentrifuge tubes.
- The tubes were centrifuged at 4000 rpm for 5 min in order to spin down the E.coli bacteria.
- Appropriate volume of ethanol as indicated by the manufacturer was added into buffer PE before starting the experiment.
- The supernatant was discarded and the bacterial pellet was resuspended in 125µl of buffer P1 which has RNAse. The contents in the tubes were mixed well using a vortex.
- The contents from both the tubes were mixed into one tube.
- About 250µl of the lysis buffer P2 was added into the tube and inverted 4 to 6 times in order to mix well.
- About 350µl of the neutralization buffer N3 was added into the same tube and inverted 4 to 6 times in order to mix well. The microcentrifuge tube was centrifuged at 13,000 rpm for 10 min.
- Without disturbing the white pellet which has formed at the bottom of the tube, the supernatant was transferred into a QIAprep spin column and centrifuged at 13,000 rpm for 30 to 60 sec. The flow through was carefully discarded.
- The spin column was washed using 500µl of binding buffer PB and centrifuged at 13,000 rpm for 30 to 60 sec. The flow through was discarded.
- The spin column was washed using 750µl of wash buffer PE and centrifuged at 13,000 rpm for 30-60 sec. The flow through was discarded.
- The tube was centrifuged for an additional 1 min in order to remove the residual wash buffer from the spin column.
- The QIAprep spin column was transferred into a fresh sterile 1.5 ml microcentrifuge tube.
- About 50µl of the elution buffer EB was added into the center of the spin column membrane. The tubes were left to stand for 1 minute and then centrifuged at 13,000 rpm for 1 min.
- Elute consists of the purified EGFP-LC3 plasmid DNA.
For determining the EGFP-LC3 plasmid DNA concentration [6]:
Materials:
- UV spectrophotometer (JASCO Analytical Instruments)
- Quartz cuvettes
- Double distilled water
- Elution buffer EB containing10mMTris-HCl with pH 8.5.
Methods:
- About 990µl of double distilled water was added into the 1ml quartz cuvette. Further about 10µl of EB was added into the same quartz cuvette and mixed well.
- The concentration was measured using the UV spectrophotometer.
- Another quartz cuvette was taken and 10µl of the purified EGFP-LC3 plasmid DNA was added. Into the same cuvette about 990µl of double distilled water was added and the DNA concentration was measured at A260nm (for DNA) and A280 nm (for protein).
- The purity of DNA concentration was determined by taking a ratio betweenOD260/OD280.
- The DNA concentration was calculated using Beer Lambert’s law (A= εlc).
For performing restriction digestion on the EGFP-LC3 plasmid DNA [6]:
Materials:
- EcoRI restriction enzyme
- Microorganism source: E.coli
- Recognition site: 5’G’AATTC3’
3’CTTAA,G5’
- 10X Restriction enzyme buffer containing 100mM Tris-HCl, 50mM NaCl, 10mM MgCl2, 0.025% Triton X-100 with pH 7.5.
- Double distilled water
- Incubator (37⁰C)
- Microcentrifuge tubes (1.5ml capacity)
Method:
- About 4µl of EGFP-LC3 plasmid DNA was taken in 1.5ml of microcentrifuge tube.
- About 1µl of 10X restriction enzyme buffer and 5µl of double distilled water was added into the tube.
- About 0.5µl of restriction enzyme EcoRI was added into the same tube.
- The tube was incubated at 37⁰C for 45 minutes.
- After incubation the tubes were kept in the freezer until they were loaded onto the 1% agarose gel.
For agarose gel electrophoresis:
Materials:
- Electrophoresis agarose powder
- TBE electrophoresis buffer containing 1M Tris, 0.9M boric acid and 0.01M EDTA
- Ethidium bromide solution (10mg/ml) (Invitrogen)
- 10X loading buffer Containing 65% (w/v) sucrose,10 mMTris-HCL (pH 7.5), 10 mM EDTA and 0.3% (w/v) bromophenol blue
- 1Kb Plus DNA ladder (Invitrogen)
- DNA molecular weight marker (1 kb plus ladder)
- Double distilled water
- Gel casting trays with specific combs
- Horizontal gel electrophoresis apparatus with power supply (ThermoScientific)
- Microwave
- UV gel documentation system
Method:
Preparation of 10X TBE buffer:
The 10X TBE buffer was prepared by dissolving 1M of Tris base, 0.9M boric acid and 0.01M EDTAin 900ml of double distilled water.The solution was mixed well and the volume was made upto 1L using double distilled water.
Assembling the gel electrophoresis apparatus:
The gel apparatus was placed on a level surface. The buffer tray was assembledwith the gel casting tray. Using the gel casting dam, the gel casting tray was sealed. Appropriate combs were placed in the slots provided.
Preparation of 1% agarose gel:
One liter of 1X TBE was prepared by mixing 100ml of 10X TBE buffer and 900ml of double distilled water. About 1g of electrophoresis grade agarose was added into a conical flask containing 100ml of 1X TBE buffer and mixed well. The conical flask was placed in the microwave and heated to melt the agarose completely. The agarose solution was cooled to about 55⁰C and 2µl of ethidium bromide (0.5µg/ml) was added and mixed well. The gel solution was poured on the gel casting tray and left to solidify.
Running the 1% agarose gel:
After confirming that the gel has solidified completely, the gel apparatus was filled with 1 XTBE buffer until the gel was completely covered. The combs were carefully removed without tearing the gel.
Three separate samples were loaded into their individual wells without spillage:
- Isolated plasmid DNA:About 5µl of the isolated plasmid DNA was mixed with 1.5µl of 10X loading buffer and 4µl of double distilled water.
- Cleaved plasmid DNA:About 5µl of the plasmid DNA which was cleaved was also mixed with 1µl of 10X loading buffer and 4µl of double distilled water.
- 1 Kb plus DNA ladder:About 0.5µl of 1Kb plus DNA ladder from Invitrogen (Fig. 1) was mixed with 7.5µl of double distilled water and 2µl of 10X loading dye.
The gel apparatus was covered with the help of the lid and the leads were connected to the power pack.The gel was placed in such a way that the DNA migrates from anode to cathode. The voltage was set 1 to 10V/cm and the gel electrophoresis was started. The progress of the DNA separation was monitored by the migration of the dyes in the loading buffer. The power supply was turned off when the bromophenol blue dye in the loading dye migrated approximately 50% of the length of the gel. The gel was placed on a UV gel documentation system to visualize and capture the gel picture. The gel picture was further used to analyze the DNA bands.
Figure & Molecular weight vs. migration graph
( hi : I don’t know but she said do the MW scale in cm and from the slop find the real molecular Weight ?!! I didn’t get it )
RESULTS
The EGFP-LC3 plasmid DNA was isolated from an overnight E.coli culture. The concentration and purity of the isolated plasmid DNA was determined using a UV spectrophotometer. The DNA concentration at A260nm was found to be 0.0165736 and the protein concentration at A280nm was found to be 0.00311872. Figure 2 shows the UV absorbance spectrum for DNA and proteins. The ratio between the ODs taken at A260nm/A280nm was calculated to determine the purity of the DNA which was found to be 5.314. Using Beer-lamberts law (A= εlc), the DNA concentration was found to be 82.868ug/ml.
Agarose gel electrophoresis was performed on the plasmid DNA and the cleaved plasmid DNA. Figure 3 shows the gel image comprising of the isolated DNA as well as the cleaved DNA. By comparing the band of the linear EGFP-LC3 plasmid DNA to the 1kb plus DNA ladder and plotting a semi-log graph using molecular weight (which is nothing but base pairs) versus the distance traveled by linear plasmid DNA, the band size was found to be 4.5kb (Fig. 4). Table 1 shows the distance travelled by the bands in the 1kb plus DNA ladder, circular plasmid DNA and the linear plasmid DNA.
DISCUSSION
For the current experiment, the QIAprep Miniprep kit from qiagen was used for isolation of EGFP-LC3 plasmid DNA. This kit presents a quick, easy and cost effective plasmid DNA isolation method which is used in molecular biology laboratory. Using this kit we were able to isolate the DNA from EGFP-LC3 plasmid successfully with a concentration of 82.868µg/ml. In order to access the purity of the isolated DNA, the absorbance of the DNA was measured using a UV spectrophotometer at A260nm for DNA and A280nm for protein. If the ratio between the two values falls in the range of 1.8 to 2.0, the DNA is usually considered as pure. However, in the present case, the ratio of ODs taken at A260nm/A280nm was found to be 5.314. This indicates that the DNA sample was contaminated with RNA or residual components. The contamination in the DNA sample might have been caused because of sample handling or operating error during the experiment [7].
One of the possible reasons for high OD260/OD280 ratio is because of the presence of degraded RNA in the sample. This could be caused due to improper washing using buffer PE during the washing step. The wash buffer allows the purification of the plasmid DNA bound to the silica membrane by removing salts and RNA. Normally, during isolation of DNA the RNA is removed from the bacterial cell by degrading it using RNase which is present in the re-suspension buffer P1 [9]. The degraded RNA is loosely bound to the silica membrane in the spin column. If the wash buffer is not applied to the silica membrane efficiently, degraded RNA will not be washed off during the washing step. When performing the elution step, the degraded RNA gets co-eluted along with the DNA, because of which the ratio between OD260/OD280 is above 2.0 in the present case. If the wash buffer contained chaotropic salts, then the degraded RNA would have been effectively washed off the membrane. Because the chaotropic salts does not allow the RNA to bind to the silica membrane strongly. There is also a possibility that there is prescence of residual ethanol in the DNA elute which can also be the reason for high OD [8-9].
The plasmid EGFP-LC3 contains a CMV promoter which allows for high levels of expression in the mammalian cells. It also has an ampicillin resistance gene and a cloning site with restriction sites that can be used to cleave the plasmid for inserting the gene of interest. For the current experiment, EcoRI restriction enzyme was used to cleave and make the plasmid linear. Agarose gel electrophoresis which is a common method used to separate and identify DNA fragments usually between 0.1 to 25kb was used to analyze the restriction digested product [10].The undigested plasmid DNA when run on the agarose gel produced 3 different bands which were supercoiled. However, the digested plasmid DNA produced 2 bands. By comparing the DNA bands with the 1kb plus DNA ladder the size of the linear plasmid EGFP-LC3 was found to be 4.7kb and the non specific band size which might be denatured plasmid DNA was found to be around 390bp.
The current report describes in detail the plasmid DNA isolation which was carried out followed by DNA quantification using UV spectrophotometer. The isolated DNA was further digested using a restriction enzyme which was run on an agarose gel to determine the size of the linear plasmid DNA.
http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=2&t=32075 (for high OD
https://biosci-batzerlab.biology.lsu.edu/Genomics/documentation/3130_NanoDrop_tips.pdf
If the ratio is above 2, then it is NOT protein contamination since that would reduce the ratio. It is more likely due to traces of phenol, alcohols or even some salts still in the sampleREFERENCES:
- “Bacterial Plasmids.” Web. 22 May 2009.
- Kado, C. I. and Liu, S. T. “Rapid Procedure for Detection and Isolation of Large and Small Plasmids.” Journal of Bacteriology (1981): 1365-1373.
- Lara, A. R. and Ramirez, O.T. “Plasmid DNA production for therapeutic applications.” Methods Molecular Biology, 824 (2012): 271-303. Print.
- Birnboim, H. C. and Doly, J. “A rapid alkaline extraction procedure for screening recombinant plasmid DNA.” Nucleic Acids Res. 7 (1979): 1513-1522.
- “QIAprepMiniprep Handbook.” 2nd Edition, 2006.
- “Plasmid extraction kit. “ Web. 2013.
- Sambrook, J. and Russell, D. W. “Molecular Cloning,” 3rd Edition, 2001, Cold Spring Harbor Laboratory Press, New York.
- “Plasmid DNA. “ Web. 6 Feb 2013, http://www.geneaid.com.
- Glasel, J. "Validity of nucleic acid purities monitored by 260/280 absorbance ratios." BioTechniques 18 (1995): 62–63.
- Lewis, Matt. “Agarose gel electrophoresis (basic method).” Web. 2001.
Add some more Reference (Research Paper. Here are few links http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217141/pdf/jbacter00274-0253.pdf
Figure 1: 1 kb plus DNA ladder run on a 0.9% agarose gel (0.7µg/lane) (Invitrogen)
Figure 2: UV absorbance spectrum for DNA and proteins. The intensity observed at A260nm was found to be 0.0165736 and A280nm was found to be 0.00311872 respectively.
Figure 3:Agarose gel electrophoresis of the isolated plasmid DNA and the cleaved EGFP-LC3 plasmid DNA. Lane 1:DNA isolated from EGFP-LC3 plasmid showing 3 supercoiled DNA bands, a, b and c;Lane 2:Cleaved EGFP-LC3 plasmid using EcoRI and a non-specific DNA band at 390bp; Lane 3:1kb plus DNAThe linear plasmid was found to be around 4.5kbp.
Figure 4: The molecular weight (which is nothing but base pairs) versus the distance traveled by the circular plasmid DNA and the linear plasmid DNA is plotted using a semi-log graph. The best fit line is drawn through the point plotted on the graph to determine the molecular weight (in base pairs) of the circular DNA and the liner plasmid DNA. The size of the linear plasmid DNA was found to be 4.5kbps.
Table 1: The distance travelled by the bands in the 1kb plus DNA ladder, circular plasmid DNA and linear plasmid DNA
