ISOLATION OF BACTERIAL PLASMID DNA THEORY

Plasmid DNA is a circular and double-stranded extra-chromosomal DNA molecule which is capable of replicating independently of the chromosomal DNA. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g. Saccharomyces cerevisiae). Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp). Plasmid Pl id size i varies i from f 1 to t over 1,000 1 000 kilobase pairs (kbp). The number of identical plasmids within a single cell can range g from one to even thousands under some circumstances. The term plasmid was first introduced by g in 1952. the Joshua Lederberg Plasmids are not considered a form of life. Plasmids as ds a are e "naked" a ed DNA a and d do not ot e encode code ge genes es necessary ecessa y to e encase case t the e genetic material for transfer to a new host.

Plasmids used to transfer foreign DNA into host bacteria, which can then produce numerous copies of foreign DNA by normal mitosis. Therefore, Th f are commonly l used d as i important t t tools t l in i genetics ti and d bi biotechnology t h l l labs, b to multiply or express particular genes. Plasmids used in genetic engineering are called vectors. Many plasmids are commercially available.

METHODS
The successful isolation of bacterial plasmid involve the following steps: Growth of the bacterial culture: Plasmids are almost always purified from liquid bacteria cultures, usually E-coli. Harvesting and lysis of the bacteria: When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. precipitated After the addition of acetate (salt or ester of acetic acid)-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution. solution Plasmid DNA yield: 20-30 g (Minipreparation); 100-350 g (Midipreparation); 500850 g (Maxipreparation); 1.5-2.5 mg (Magapreparation); 7.5-10 mg (Gigapreparation). (Gigapreparation) Purification of plasmid DNA: Addition of phenol/chlorofrom can dissolve and denature proteins.

METHODS: Growth of the bacteria:

Liquid q media are used for g growth of p pure batch cultures while solidified media are used widely for the isolation of pure cultures. A selective medium is one which has a component(s) added to it which will inhibit or p prevent the g growth of certain types yp or species p of bacteria and/or promote the growth of desired species. LB broth (Lysogeny broth), a nutritionally rich medium used for the growth of bacteria. medium, bacteria
Mediu m Name LB Miller Broth LB Lennox Broth Applications E.coli growth and propagation; plasmid DNA and protein production E.coli growth and propagation; plasmid DNA and protein production Contents 1% peptone, 0.5% yeast extract, and 1% NaCl 1% peptone, 0.5% yeast extract, and 0.5% NaCl

METHODS : Harvesting and lysis of the bacteria

Alkaline Alk li M th d Method Boiling Method CsCl Method LiCl Method Qiagen Method SDS Method TELT Method

ALKALINE LYSIS
Alkaline [base, especially soluble bases, pH = >7; basic, ionic salt of an alkali metal, e g lithium (Li), e.g. (Li) sodium (Na), (Na) potassium (K)], (K)] lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or other cell components such as proteins. Bacteria containing the plasmid of interest is first grown, grown then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl sulfate (SDS) [an anionic surfactant used in many cleaning and hygiene products], and a strong base sodium hydroxide. The detergent breaks the membrane's phospholipid bilayer and the alkali denatures proteins involved in maintaining the structure of the cell membrane.

Procedure: using grown Bacterial Culture

Materials: Solutions
Solution 1: 50 mM glucose 10 mM EDTA pH 8.0 Solution 2: 1% SDS 0.2 N NaOH S l i Solution 3: 3 M K+ 5 M Acetate TE 10 mM Tris-HCl pH 8.0 1 mM M EDTA per 500 ml: 9 ml 50% glucose 10 ml 0.5 M EDTA pH 8.0 per 500 ml: p 50 ml 10% SDS 100 ml 1 N NaOH per 500 ml: l 300 ml 5 M Potassium Acetate 57.5 ml glacial acetic acid per 100 ml: 1 ml 1 M Tris-HCl pH 8.0 0 5 ml 0.5 l 0.5 0 5 M EDTA pH H 8.0 80 Add H2O to 500 ml. Add H2O to 500 ml. Add H2O to 500 ml. Add H2O to 500 ml.

25 mM Tris-HCl T is HCl pH 8.0 8 0 12.5 12 5 ml 1 M Tris-HCl T i HCl pH 8 8.0 0

Optional: RNAse can be added to TE at final concentration of 20 ug/ml.

Mi icropipett ter tip

Materials: Supplies

Vortex mixer

Micr rocentrifu uge

Microcentrifuge tubes

Procedure: using grown Bacterial Culture

1 Fill a microcentrifuge tube with saturated bacterial culture grown in LB 1. broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel. 2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. prep Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel. 3. Add dd 0.2 ml l ice-cold ld Solution l 1 to cell ll pellet ll and d resuspend d cells ll as much h as possible using disposable transfer pipet. Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity. activity

4. Add 0.4 ml Solution 2, cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes. Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands. strands 5. Add 0.3 ml ice-cold Solution 3, cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes. Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the h SDS S S from f solution, l i along l with i h the h cellular ll l d b i The debris. h E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution. 6. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh microcentrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.

This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet. 7. Fill remainder of f centrifuge f tube with isopropanol. Let tube sit at room temperature for 2 minutes. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants. 8 Centrifuge tubes for 5 minutes. 8. minutes A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel. This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period. 9. Add 1 ml of ice-cold 70% ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel. Ethanol helps to remove the remaining salts and SDS from the preparation.

10. Allow tube to dry for ~5 minutes. Add 50 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored t d indefinitely i d fi it l in i the th freezer. f Reference: Department of Molecular and Cellular Biology The University of Arizona October 6, 1997 Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu

Procedure: with growing BAC cultures using QIAGEN KIT

1. Pick a single BAC colony and inoculate a starter culture of 5 ml LB 1 mediumcontaining the appropriate antibiotic. 2. Inoculate 0.5 ml pre-culture into 100 ml selective LB medium. Grow at 37C for 14 hours with vigorous g shaking g( (250 rpm). p ) 3. Divide the cells into two 50 ml tubes, and harvest the cells by centrifugation at 4500 x g for 20 min. 4 Resuspend each bacterial pellet in 10 ml Buffer P1. 4. P1 Ensure that RNase A (100 g/ml) has been added to Buffer P1. 5. Add 10 ml Buffer P2 to each tube. Mix thoroughly and gently by inverting 46 4 6 times, and incubate at room temperature for 5 min. Check Buffer P2 before use for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37C. y mix by y g gently y 6. Add 10 ml chilled Buffer P3 to each tube. Immediately inverting 46 times, and incubate on ice for 15 min. 7. Centrifuge at 20,000 x g for 30 min at 4C. Remove supernatant containing plasmid DNA promptly. 8. Centrifuge the supernatant again at 20,000 x g for 15 min at 4C. Remove supernatant containing plasmid DNA promptly.

9. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow. 10. Pool the two supernatants from step 8. Apply the sample to the QIAGEN-tip and allowit to enter the resin by gravity flow. 11. Wash the Q QIAGEN-tip p with 2 x 10 ml Buffer Q QC. 12. Elute DNA with 5 x 1 ml Buffer QF, prewarmed to 65C. Prewarming the elution buffer may help to increase yields. Eluting in 5 q of 1 ml instead of 1 aliquot q of 5 ml p prevents cooling g of the elution aliquots buffer. 13. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA. Mix and centrifuge g immediately y at 15,000 , x g for 30 min at 4C. Carefully decant the supernatant. pellet with 2 ml of room-temperature p 70% ethanol and 14. Wash the DNA p centrifuge at 15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet. y the p pellet for 510 min, , and redissolve the DNA in a suitable 15. Air-dry volume of buffer (e.g., TE, pH 8.0, or 10 mM TrisCl, pH 8.5).

Preparation of Genomic DNA from Bacteria - using Phase Lock GelTM

(Modified from Experimental Techniques in Bacterial Genetics, Jones and Bartlet, 1990)

Materials: TE buffer 10% (w/v) sodium dodecyl sulfate (SDS) 20 mg/ml proteinase K phenol\chloroform (50:50) isopropanol 70% ethanol 3M sodium acetate pH 5.2 Procedures: 1. Grow E. coli culture overnight in rich broth. 2. Transfer 2 ml to a 2-ml micro centrifuge tube and spin 2 min. 3 Decant the supernatant. 3. supernatant 4. Drain well onto a Kimwipe.

5. Resuspend the pellet in 467 l TE buffer by repeated pipetting. 6. Add 30 l of 10% SDS and 3 l of 20 mg/ml proteinase K, mix i , and d incubate i b 1 hr h at 37 C. C 7. Add an equal volume of phenol/chloroform and mix well but very gently to avoid shearing the DNA by inverting the tube until the phases are completely mixed. CAUTION: Phenol causes severe burns, wear gloves goggles, and lab coat and keep tubes capped tightly. 8. Carefully transfer the DNA/phenol mixture into a Phase Lock GelTM tube (green) and spin at 12,000 RPM for 10 min. 9. Transfer the upper pp aqueous q phase to a new tube and add an p equal volume of phenol/chloroform. 10. Again mix well and transfer to a new Phase Lock GelTM tube and spin 10 min. 11. Transfer the upper aqueous phase to a new tube. 12. Add 1/10 volume of sodium acetate. Mix. 13 Add 0.6 13. 0 6 volumes of isopropanol and mix gently until the DNA precipitates.

14. Spool DNA onto a glass rod (or Pasteur pipet with a heatsealed end). 15. Wash DNA by dipping end of rod into 1 ml of 70% ethanol for 30 sec. 16. Resuspend DNA in at least 200 l TE buffer. Complete resuspension may take several days. Store DNA at 4 C short term, -20 or -80 C long term. 17. After DNA has dissolved, , determing g the concentration by y measuring the absorbance at 260 nm.

Bacterial DNA Preparation, Jeff Lawrence 1. Grow 5 ml culture overnight. 1 overnight Pellet 1.5 1 5 ml culture in a microcentrifuge tube for 3 min. Remove supernatant with a vacuum aspirator and resuspend pellet in 1 ml TE: 10 mM Tris, pH 8.0 (1 mL 1 M Tris, pH 8.0) 1 mM EDTA (200 L 500 M EDTA, pH 8.0) (99 mL ddH2O) 2. Microfuge as above, remove supernatant, and resuspend pellet in 400 L NET: 100 mM NaCl (2 mL 5 N NaCl) 50 mM EDTA (10 mL 500 mM EDTA, pH 8.0) 50 mM Tris, pH 8.0 (5 mL 1 M Tris, pH 8.0) (83 mL ddH2O) 3. Add 25 l 20 mg/ml g/ Lysozyme y y in NET. Incubate on ice for 10 min. 4. Add 30 l 10% SDS and incubate on ice for an additional 2 min. 5. Place tubes at 65 for 15 min then cool to room temperature. 6. Add 400 l Buffered phenol. Mix well by vortexing and microfuge for 15 min at 4. Transfer supernatant to a new tube. Avoid taking any material at the interface.

7. Add 400 l Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking and vortexing and microfuge for 2 min. Transfer supernatant to a new tube. tube 8. Add 850 L 95% Ethanol and mix by inversion. DNA should precipitate p p immediately. y Microfuge g for 3 minutes. 9. Discard supernatant. Begin Optional Rnase section Begin, 10. Dry in vacuum desiccator for 4 min or until just dry. Do not overdry. Resuspend pellet in 200 L TE. As an option, RNA may be removed as described below. 11. Add 8 L 5 N NaCl and 5 L 2 mg/ml RNase (boiled 10 min before first use). use) Incubate for 30 min at 37 37. 12. Add 500 L 95% ethanol and mix by inversion. Microfuge for 3 minutes. Discard supernatant.

End, Optional Rnase section 13. Add 1 mL 70% ethanol. Rinse pellet well, dislodging it from the bottom of the tube. Microfuge for 2 min. Discard supernatant. 14. Dry in vacuum desiccator as above. Resuspend pellet in 100L TE. Yield is 0.5-2.0 mg/ml.