Purification and Characterization of a DNA Plasmid Part A CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008 INTRODUCTION DNA Plasmids. A plasmid is a small double-stranded, circular DNA molecule that replicates independently of chromosomal DNA. A plasmid must contain an origin of replication, and may contain promoters, antibiotic resistance genes, other coding sequences and polylinker regions. (A polylinker is a region of DNA within a cloning vector that contains numerous recognition sites for a wide variety of restriction enzymes. Usually the gene of interest is embedded into the plasmid in the polylinker region.) Plasmids can be used as cloning vectors for the production of DNA, RNA or ultimately for protein overexpression. The plasmid to be purified in this study is called pyp (Figure 1a) because the gene that encodes for a mutant of of yeast phenylalanine trna was cloned into the parent vector. The pyp plasmid was derived from pdefend2 (Figure 1b), which was in turn derived from the DNA plasmid puc19 (not shown). Each of these plasmids contains a gene that confers resistance to the antibiotic ampicillin. The parent plasmid pdefend2 also contains a promoter for T7 RNA Polymerase followed by a polylinker. A B Figure 1. Schematic of DNA plasmids (A) pyp and (B) pdefend2. Arrows indicate the direction of transcription of the DNA. Note: Color scheme in the DNA sequence was not intended to match that in the depiction of the plasmid. Cleavage of pdefend2 with the restriction enzymes Bsa I and Bst I within the polylinker region allow for insertion of the sequence encoding a mutant of yeast trna Phe. Illustrations were generated by Professor Loren D. Williams (Georgia Tech). 1
Alkaline Lysis. There are many different protocols for isolating plasmid DNA from bacterial cultures. One common and reliable chemical procedure, which will be used here, is known as the alkaline lysis method. In the alkaline lysis method, bacteria are grown in Luria Bertani (LB) broth and are pelleted by high-speed centrifugation. The pellet is suspended in an alkaline solution (NaOH) to lyse the bacteria and to denature proteins and DNA. Then the solution is neutralized in potassium acetate. Bacterial proteins aggregate and precipitate, entangling large chromosomal DNA fragments, which are too large to renature. The presence of sodium dodecyl sulfate (SDS) also helps to form micelles around any precipitant. The plasmid DNA is relatively small, does renature and remains free in solution. Following centrifugation, the plasmid DNA (in the supernatant) is further purified using a commercial chromatographic resin. Plasmid Purification. The Promega Wizard Plus Midiprep kit is designed for the efficient and easy purification of DNA plasmids. Most preparative DNA resins are commercially available, but are proprietary. Purification is generally based on either the reversible interaction of DNA with silicabased resins or ion-exchange resins. The resin described here is believed to be silica-based. The DNA will form a tight interaction with the resin until it is eluted with a "warm" solvent as described. GOAL In this two-week study, students will isolate and purify the pyp DNA plasmid from E. coli and confirm the identity by restriction mapping. Part 1 of this study is described here. After any purification, experiments must be performed to verify the success of the purification. In this case, the purified DNA plasmid will be digested with restriction enzymes, and the restriction fragments will be separated on an agarose gel. Analysis of the agarose gel via restriction mapping will be performed to provide evidence for confirming the identity of the DNA plasmid purified. For this experiment, you will receive a pellet of E. coli cells that have been engineered to contain the pyp DNA plasmid. The specific aim of this experiment is to (1) purify DNA from the E. coli cells and (2) determine the concentration of DNA purified. Using the volume of DNA recovered and its concentration ( g/ml), one can calculate the actual yield of DNA in mg and compare that value with the expected recovery published by Promega. The purified DNA will be stored at -20 C until further use in a subsequent laboratory session. MATERIALS AND REAGENTS Promega Wizard Plus Midiprep DNA Purification System Resuspension Buffer: 50 mm Tris-HCl, ph 7.5; 10 mm EDTA; and 100 g/ml RNase A (CAUTION: Use caution when handling this reagent to avoid contaminating the laboratory!) Lysis Buffer: 200 mm NaOH and 1% (w/v) SDS Neutralization Solution: 1.32 M Potassium Acetate, ph 4.8 Midiprep Resin (proprietary) in 7 M Guanidine HCl Column Wash Buffer: 80 mm Potassium Acetate; 8.3 mm Tris-HCl (ph 7.5); 40 M EDTA; and ~50% Ethanol Disposable purification columns 2
General Reagents LB Miller Broth and Agar 100 mg/ml Ampicillin (Sigma) Pellet of E. coli cells 50-100 mm Tris-HCl, ph 8 50-mL Oak Ridge Centrifuge Tubes Petri Dishes Inoculating Loops Shaking Incubator 37 ºC Incubator Ultracentrifuge Shimadzu UV-1601 UV-visible spectrophotometer Ear I restriction enzyme with 10X NEBuffer 1 (supplied with enzyme; New England Biolabs) EXPERIMENTAL PROCEDURE NOTE: You must wear gloves throughout this lab to protect your DNA/RNA. Please use autoclaved tips, water, etc. for all solutions used in the molecular biology steps. Production of pyp Plasmid DNA - Performed by the TA These details are provided here so that each student will know how the DNA pellet was generated. Day 1 Day 2 1. Prepare enough of the LB (Miller) agar solution according to the manufacturer's instructions to accommodate ~3 plates. You only need one, but the others will serve as a backup. 2. Autoclave the agar solution then allow it to cool to ~50 ºC. 3. To the agar solution, add 1 L of 100 mg/ml ampicillin (dissolved in sterile water) for every ml of agar. Pour the agar onto a sterile petri dish and allow the agar to solidify. 4. Use the sterilized loop to streak the surface of the agar with E. coli cells that have been transformed with the DNA plasmid of interest. Aliquots of these cells have been stored at -70ºC. Leave the plate inverted at 37ºC overnight. 5. Prepare LB (Miller) media (broth) according to the manufacturer's instructions. Autoclave 500 ml of media in each of six 2L flasks; this will generate enough cells to accommodate 30 replicates of 100 ml each. Be sure to also prepare a 50-mL starter culture in a 250 ml flask. Keep the flasks covered at all times to maintain sufficient sterility. Store the media at 2-8ºC until further use. 1. Early in the morning, isolate a single colony from the freshly streaked selective plate and inoculate a 5 ml LB/AMP starter culture. Incubate the starter culture in a shaking incubator with vigorous shaking (Setting #4 on shaking incubator) for ~8 hours at 37 ºC. 2. In the afternoon (i.e. just before inoculating the large cultures), add 0.5 ml of 100 mg/ml ampicillin (or the appropriate antibiotic) to each flask of LB media. (Do not leave the antibiotic in the media for long periods before the inoculation in the next step.) 3. At the end of the 8-hour shaking incubation, add 0.67 ml of the starter culture to each 500 ml sterile media flask. Grow the cells to late log phase in a shaking incubator with vigorous shaking at 37ºC for 12-16 hours. 3
Day 3 1. Pellet the E. coli cells by centrifugation at 6000 x g for 15 minutes at 4ºC in 250 ml Sepcor bottles. Discard the supernatant and resuspend the cells in buffer at a neutral ph. 2. Transfer the appropriate amount of cells to 50 ml Oak Ridge tubes. Each student will receive one of these tubes, so each tube should contain cells corresponding to 100 ml of the original culture. 3. Centrifuge the cells at 6000 x g for 15 minutes at 4ºC. Discard the supernatant and store the pellet of bacteria at -20ºC until further use. Purification of pyp - Performed by the Student 1. Keeping the pellet on ice, resuspend the bacterial pellet in 3 ml of Resuspension Buffer by repeated pipetting. Be sure to fully disperse the pellet. 2. Lyse the bacterial cells by adding 3 ml of Lysis Buffer to the suspended pellet. Close the container of Lysis Buffer immediately to prevent contamination. (Note: Before using the Lysis Buffer, you may need to warm it to 37ºC to insure that the SDS is in solution.) Mix the suspended pellet gently but thoroughly, by inverting 4-6 times. Incubate at room tempertature for no more than 5 minutes. Do not vortex your sample; vortexing will shear genomic DNA! 3. Add 3 ml of chilled Neutralization Buffer to the pellet mixture. Mix immediately but gently by inverting 4-6 times. Your solution may become biphasic with a cloudy precipitate and a clear phase. 4. Centrifuge at ~14,000 x g (11,000 rpm) for 30 minutes at 4ºC. Your supernatant will be clear after centrifugation. Transfer the supernatant (which contains the plasmid DNA) to 10 ml of Midiprep resin. Mix by swirling. This allows the DNA to bind to the resin in batch mode. Discard the pellet. 5. Place the column tip (labeled with your initials) into the vacuum manifold. Pour the DNAresin slurry into the column. Apply vacuum to pack the slurry into the column. Once the "flow-through" has eluted, break the vacuum. Do not allow the resin to dry out! 6. Wash the column once with 15 ml of Wash Buffer. Apply vacuum to pull the wash buffer through the column. Repeat this step. Meanwhile, start heating the sterile water aliquots. 7. Dry the resin some by applying the vacuum for no more than 30 seconds after all solution has eluted from the column, then break the vacuum. 8. Cut the reservoir off of the column with scissors and transfer the column to a 1.5 ml microcentrifuge tube with its lid cut off. Be sure to balance each sample by weight. Centrifuge the column at 10,000 x g (Setting "7" on the Fisher Marathon Micro A centrifuge) for 2 minutes at room temperature to remove any residual wash solution. Then, transfer the column to a new 1.5 ml microcentrifuge tube. (Note: The lid to the microcentrifuge may not fit securely with these tubes in place. Screw on the lid as evenly as possible.) 9. Add 0.3 ml of pre-heated (65-70ºC) autoclaved di water (i.e. nuclease-free water) to the column for one minute. Elute the DNA by centrifuging the column at 10,000 x g (Setting "7") for 20 seconds. Discard the column. 4
10. Finally, centrifuge the DNA sample at 10,000 x g (Setting "7") for 5 minutes to pellet any fine particles of resin that may have eluted with the DNA. These fine particles may not be easily visible. Transfer at least 250 L of the supernatant to a fresh tube labeled with your initials and "pyp". Analysis of Purified pyp DNA Plasmid 11. Add 50 L of the purified pyp DNA solution to 950 L of 100 mm Tris-HCl at ph 8 directly in a cuvette. 12. Measure the absorbance of the DNA solution at 260 nm using Plastibrand cuvettes since they are transparent from 220-900 nm. Record the absorbance measurement in your lab notebook. 13. As described by Sambrook and coworkers in the reference below, determine the concentration (in g/ml) of the purified DNA sample as follows: [DNA] ( g/ml) = (A260) (50 g/ml) (Dilution Factor) 14. Store your purified DNA at -20 C until further use. Reference Sambrook, J., and Russell, D.W. (2001) Commonly Used Techniques in Molecular Cloning, in Molecular Cloning: A Laboratory Manual 3 rd ed., pp A8.20-A8.21, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 5