* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Plasmid Isolation Using Alkaline Lysis
Metagenomics wikipedia , lookup
DNA paternity testing wikipedia , lookup
Designer baby wikipedia , lookup
DNA barcoding wikipedia , lookup
DNA methylation wikipedia , lookup
Epigenetics wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Epigenetic clock wikipedia , lookup
DNA sequencing wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Genetic engineering wikipedia , lookup
Microevolution wikipedia , lookup
Point mutation wikipedia , lookup
DNA profiling wikipedia , lookup
Cancer epigenetics wikipedia , lookup
DNA polymerase wikipedia , lookup
SNP genotyping wikipedia , lookup
Primary transcript wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Genomic library wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Non-coding DNA wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Epigenomics wikipedia , lookup
Helitron (biology) wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Molecular cloning wikipedia , lookup
DNA vaccination wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
DNA supercoil wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Experiment 3 for CSS451 Plasmid Isolation Using Alkaline Lysis Plant Biotechnology Resource & Outreach Center, Michigan State University Plasmid Isolation Protocol 1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial colony. The tube was incubated at 37 ˚C overnight with vigorous shaking at 360 rpm. 2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature. 3. Discard the supernatant. 4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet up and down or vortex as necessary to fully resuspend the bacteria. 5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex. 6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex. 7. Centrifuge at 15,500 x g for 30 minutes at 4C. 8. Recover resulting supernatant. 9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated gentle inversion. Do not vortex. 10. Centrifuge at 15,500 x g for 30 minutes at 4C. 11. Removal of resulting supernatant. The pellet is plasmid DNA. 12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate. 13. Add ddH2O or TE to dissolve the pellet. After addition of 2ul RNase A (10mg/ml), the mixture was incubated for 20 minutes at room temperature to remove RNA. Note: 1. Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage. 2. Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip. 3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells. 1 4. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). Three things happen during this stage: a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo and toothpaste. b. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA. c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained. 5. Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things: a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single stranded DNA (ssDNA). b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt. c. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from your plasmid DNA. Now that you've made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet. 6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to precipitate. This will place your DNA in the pellet. 7. Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate. 8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution. DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you'll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification. 2 Isolation of the Plasmid after Alkaline Lysis The plasmid "miniprep " method is useful for preparing partially purified plasmid DNA in small quantities from a number of transformants. It relies on an alkaline SDS lysis to free the plasmid DNA from the cell, leaving behind the E. coli chromosomal DNA with cell wall debris. The protocol described involves three basic steps: growth of bacteria and amplification of the plasmid; harvesting and lysis of the bacteria; and purification of the plasmid DNA. These purification procedures exploit in one way or another the two major differences between Escherichia coli DNA and plasmid DNA: 1. The E. coli chromosome is much larger than the DNA of plasmids used as vectors. 2. The bulk of E. coli DNA extracted from cells is obtained as broken, linear molecules. By contrast, most plasmid DNA is extracted in a covalently closed, circular form. The purification protocol therefore involves a differential precipitation step, in which the long strands of E. coli DNA, entangled in the remnants of lysed cells, are preferentially removed. Because each of the complementary strands of plasmid DNA is a covalently closed circle, the strands cannot be separated (without breaking one of them) by conditions such as exposure to mild alkali (up to pH 12.5), which break most of the hydrogen bonds of DNA. Closed circular molecules regain their native configuration when returned to neutral pH.E. coli remains in the denatured state. This method provides enough purified plasmid DNA for sequencing. 5 ml LB medium were inoculated with a single bacterial colony. The tube was incubated at 37psy176 C overnight with vigorous shaking. 4.5 ml of the culture were centrifuged for 20 minutes at 3500 rpm at 4psy176 C. The remainder of the overnight culture was stored at 4psy176 C. The medium was removed, leaving the bacteria pellet as dry as possible. The pellet was resuspended in 150 l ice-cold Lysis buffer I (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) and stored for 5 minutes at room temperature. After adding 300 l freshly prepared and mixing by inversion, the mixture was incubated for 5 minutes on ice. Now 225 l ice-cold 3M KAc/5M HAc (pH 6.0) were added and mixed gently. The tube was stored on ice for 5 minutes, precipitating the chromosomal bacteria DNA. After centrifugation for 15 minutes at 13000 rpm at 4psy176 C, the supernatant was transferred to a fresh tube. Proteins were removed by vortexing with 400 l phenol (see section phenol), adding 300 l Sevac and centrifuging for 2 minutes at 13000 rpm. 500-600 l of the aqueous layer were removed and mixed with 1 ml ethanol to precipitate the DNA (see section EtOH). After incubating for 5 minutes at room temperature, the Eppendorf tube was centrifuged for 10 minutes at 13000 rpm at 4psy176 C. The 3 supernatant was removed, the pellet washed with 70% ethanol and recentrifuged. After vacuum drying, Solution I (Lysis buffer I): 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. Store at 0˚C a. 10ml 500mM Glucose b. 2ml 500mM EDTA pH 8.0 c. 2.5ml 1M Tris pH 8.0 d. 85.5ml H2O e. Autoclave and store at 4°C Solution II (Lysis buffer II): Freshly prepared 0.2 N NaOH, 1% SDS. Store at room temperature (RT) Isopropanol: Stored at -20 0˚C Solution III (Lysis buffer III): 3M KOAc, pH 6.0 a. 60ml 5M potassium acetate (49.07g potassium acetate in 100ml H2O) b. 11.5ml glacial acetate c. 28.5ml H2O 4