MISS :Salsabeel AL Jou jou Plasmid DNA Isolation MISS :Salsabeel AL Jou jou

Purpose: To understand the basic procedures involved in the isolation process of plasmid from bacteria. To be understood the differences between two methods of plasmid isolation. To estimation Plasmid DNA quality and quantity. To visualization separating DNA with gel electrophoresis by staining with ethidium bromide. To use plasmid in transformation after isolated from bacterial cells.

Plasmids: vehicles of recombinant DNA Bacterial cell Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate

Plasmid DNA isolation Plasmid DNA isolation requires separation of this DNA from the chromosomal DNA in the bacterial cell as well as from the polysaccharides, lipids and proteins that constitute the cell.

Principle: The various plasmid isolation techniques that are currently in use can be divided into three phases: growth of microorganisms that contain the plasmid Harvesting , resuspension and lysis of bacterial cells. Separate plasmid DNA from contaminants . Purification of plasmid DNA by separating it from proteins and chromosomal DNA.

Methods for Plasmid Isolation Depending on amount of plasmid to be isolate Miniprep Maxiprep Depending on the principle there are several method to isolation plasmid SDS-alkaline denaturation Rapid boiling method

In our lab :- we used miniprep method (promega kit). purification plasmid by solid phas purificatin (with used silica based column ) learns Principle of SDS-alkaline denaturation.

Principle of the SDS-alkaline denaturation method

Overnight Culture Suspension Pick a single colony and inoculate in 5 ml of LB containing 20 mg/l ampicilin Incubate overnight at 37oC The next day: Centrifuge 1.5 ml of broth containing cells in a tube Discard supernatant

Resuspension solution (soln1) Resuspension of the bacterial pellet in buffer containing glucose ,Tris ,EDTA Glucose : Give osmotic shock that lead to rupture of cell wall and membrane The ethylene diamine tetraacetate (EDTA) chelates divalent metals (primarily magnesium and calcium) then inhibits Dnases activity of cellular enzymes.

Tris is buffer substance In addition to the above listed components, the resuspension solution can also contain RNase enzyme in order to break down ribonucleic acids.

alkaline lysis solution (soln2) Mix this by gentle inversion and incubate on ice . This solution contain: 1-SDS 2-NaOH

a. SDS (sodium dodecyl sulfate) Dissolves membranes (disintegrates the lipid structure of the cell membrane) Binds to and denatures proteins b. NaOH denatures both plasmid and chromosomal DNA to single strand . Chromosomal DNA separate completely into single strand but plasmid not fully separate and remain entangled after denaturation.

solution 3 effects: Potassium acetate / acetic acid solution A-Acetic acid :Neutralizes NaOH plasmid DNA is allowed to renature. Sheared cellular DNA remains denatured as single stranded DNA (ssDNA). The ssDNA is precipitated, since large ssDNA molecules

Since plasmid DNA is small circular and supercoiled, whereas genomic DNA is too long to reaneal properly and remain single strand .

Potassium acetate: Converts soluble SDS to insoluble PDS the potassium salt of SDS is insoluble, so the protein and detergent precipitate and aggregate, which assists in the entrapment of the high-molecular-weight chromosomal DNA.

Separate plasmid DNA from contaminants Separate plasmid DNA from contaminants by centrifugation Supernatant contains: - Plasmid DNA - Some cellular constituents Sediment contains: - PDS - Lipids - Proteins - Chromosomal DNA NOTE: The soluble plasmid DNA (supernatant) is ready to be further purified.

alkaline lysis Alkaline conditions denature DNA

purify plasmid DNA using silica-based columns (Promega kit)

The PureYield™ Plasmid Miniprep System isolates high-quality plasmid DNA for use in eukaryotic transfection and in vitro expression experiments. The system provides a rapid method to purify plasmid DNA using a silica-membrane column. Plasmid DNA can be purified in less than 10 minutes, depending on the number of samples processed, greatly reducing the time spent compared to silica resin or other membrane column methods. The PureYield™ Plasmid Miniprep System is designed to purify 1.5–7.5μg of plasmid DNA .

This system can be used to isolate any plasmid from E. coli hosts but works most efficiently when the plasmid is up to 20,000bp in size. Note: For best results, use plasmids that are 10,000bp or less. The protocol presented in this Technical Bulletin describes isolation of plasmid DNA from E. coli.

Plasmid yield will vary, depending on : 1. a number of factors, 2. culture volume, 3. plasmid copy number, 4. type of culture medium 5. bacterial strain used.

Component of kit: (30–37ºC ) Cell Lysis Buffer (CLC) (Blue. • Neutralization Solution (NSC). (store at 4-8 c) • Endotoxin Removal Wash (ERB). • Column Wash Solution (CWC). • Elution Buffer (EBB). Tris-HCl (pH 8.5) , EDTA. • PureYield™ Minicolumns. • PureYield™ Collection Tubes.

Before you begin: dilute the Column Wash Solution (provided) with 95% ethanol.

PureYield™ Plasmid Miniprep System Protocol : Centrifugation Protocol. vacuum Protocol. Alternative Protocol for Larger Culture Volumes.

Protocol :- (Centrifugation Protocol.) Check the Cell Lysis Buffer to be sure that components have not precipitated during shipping. If precipitation has occurred, resuspend the buffer by incubating the bottle at 30–37ºC for 30 minutes and mixing by inversion. *Note:- We recommend the use of LB medium. If you are using other growth media, pellet the cells and resuspend the cell pellet in TE bufer or water prior to lysis. *استخدام kit promega وفي حالة وجود الخلايا البكتيرية في ميديا L.B فإننا نستخدم الخلايا مباشرة , ولكن لو استخدمنا ميديا أخرى نقوم بعمل حصاد للخلايا عن طريق centrifug ثم نعمل لها تعليق اما في ماء أوTE buffer

Perform the following procedure at room temperature. 1. Transfer 600μl of bacterial culture grown in LB medium to a 1.5ml microcentrifuge tube. 2. Add 100μl of Cell Lysis Buffer, and mix by inverting the tube 6 times. *( The solution should change from opaque to clear blue, indicating complete lysis) 3 . Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting the tube.

Note:- The sample will turn yellow when neutralization is complete, and a yellow precipitatewill form. Invert the sample an additional 3 times to ensure complete neutralization. 4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes. 5. Transfer the supernatant (~900μl) to a PureYield™ Minicolumn.

Note:- Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette. 6. Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds. 7. Discard the flowthrough, and place the minicolumn into the same PureYield™ Collection Tube. 8. Add 200μl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge

9. Add 400µl of Column Wash Solution to the minicolumn 9.Add 400µl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds. 10. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30µl of Elution Buffer directly to the minicolumn matrix. Let stand for 1 minute at room temperature. Notes: Nuclease-free water at neutral pH can also be used to elute DNA. 11. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.

If you use promega kit : 100μl of Cell Lysis Buffer Harvest cells by centrifugation , if cells was cultured in other growth media If the cells was cultured in L.B media you can tack 600M and put cell lyses directly , without harvesting by centrifuge *Spin ~5,000 rcf Supernatant (clear)‏ *Bacteria harvest by centrifuge was dependent cell size but , general using 8000 rpm /5 min or 5000 rpm / 10 min *Use of high RPM will disrupt the cells and may make the resulting pellet unusable for further use Pelleted cells 100μl of Cell Lysis Buffer resuspend the cell pellet in TE bufer. Discard supernatant or water prior to lysis

After add lysis solution and neutralization buffer the cell will lysis , protein & lipid will precipitate and denaturation of DNA and plasmid but plasmid returned by neutralization buffer.

(Plasmid DNA ,Soluble cellular constituents) Supernatant (Plasmid DNA ,Soluble cellular constituents) pellet Protein , lipid, chromosomal DNA) Flowthrough:its liquid in the collection tube have contaminant (lipid , protein , and anything unless plasmid ) after centrifuge step plasmid attached with silica , but not completely pure so we add endotoxin and washing solution to remove all things in silica unless plasmid Finley we have pure plasmid attached with silica but we have plasmid only so we add elution buffer and after centrifuge the we will get pure plasmid in our apendrof

Inverted until the color turn to yallow Centrifuge 3min Add 600M cell culture add 100M lysis solution Inverted and add 350M neutralization solution Transfer supernatant to Minicolumn Centrifuge 15s Centrifuge 15s Flowthrough* Flowthrough:its liquid in the collection tube have contaminant (lipid , protein , and anything unless plasmid ) In this step plasmid attached with silica , but not completely pure so we add endotoxin and washing solution to remove all things in silica unless plasmid Finley we have pure plasmid attached with silica but we have plasmid only so we add elution buffer and after centrifuge the we will get pure plasmid in our ependrof Discard the flowthrough, and place the minicolumn into the same Collection Tube 200μl of Endotoxin Removal Wash 400µl of Column Wash Solution Note:- we will use on column only

Centrifuge 30s Transfer the column in apendrof tube Centrifuge 30s Add 30µl of Elution Buffer Pure plasmid

General Instructions in procedure DNA is very sensitive to mechanical stress, therefore shearing forces caused by mixing/vortexing or fast pipetting must be avoided as soon as cell lysis occurs. All mixing steps during and after cell lysis should be performed carefully by inverting the tubes several times (8-10 fold). Gloves should be worn in order to prevent contamination with Dnases Autoclaved (DNase-free) buffer solutions, tubes and tips should be used. For long-term storage, plasmid DNA should be frozen in aliquots of storage TE buffer. Repeated thawing and freezing of DNA should be avoided.

Finally(practical quize):- 1. Each student has to extracted plasmid alone by follow the promega Kit. 2. Check the quality of your extracted plasmid in nanodrope instrument. 3. Preaper1% Agarose gel , and loading your sample.

Good Luke