Himanshu chaudhry

1. PLASMID ISOLATIONANDPURIFICATION

2. PRINCIPLE
Plasmid for routine molecular cloning method is often purifiedby one of the following methods :-
Alkaline lysis method
Boiling method
Lithium chloride base
The purity of plasmid isolated by these above methods depends on how efficiently a method can separate plasmid DNA from genomic DNA. Most of these plasmid purification methods allow the preferential recovery of circular plasmid DNA over linear chromosomal DNA.
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3. ALKALINE LYSIS method
Alkaline lysis method is one of the most commonly used method for lysis bacterial cells prior to plasmid purification. It has four basic steps :-
1.Resuspension : Harvested bacterial cells are resuspended by usingsolutionIcontains
EDTA (ethylene diaminetetra-acetic acid)and Tris-CL.
EDTA chelates the magnesiumand calcium ions
Tris-CL maintains pH.
2 . LYSIS : Cells are lysis with alkaline solution II contains NaOH and SDS (sodiumdodecyl sulfate).
NaOH --denatures the chromosomal and plasmid DNAs as well as proteins.
SDS --solubilizes the phospholipids and protein components of the cell membrane, leading to lysis
and release of the cell membrane.
3.NEUTRALIZATION:The lysate is neutralized by addition ofsolution III of acidic potassium acetate. The high salt
concentration causespotassium dodecyl sulfate (KDS) to precipitate and denatured proteins,
chromosomal DNA andcellular debris are co-precipitated ininsoluble.
.
4. CLEARING OF LYSATES: Precipitated debris is removed by either high speed centrifugation or filtration, producing cleared lysates
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4. continue.
Step s
and
procedure
in
ALKALINE LYSISMETHOD
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5. .
Continue..
Harvest cells by centrifugation
Spin ~5,000 rcf
Supernatant (clear)
E. coli culture
(cloudy)
Pelleted cells
Discard supernatant
Residual media may interfere with downstream steps
Resuspend cells in buffer
Thoroughly resuspend cells, making sure that no
clumps remain. P1 buffer contains:
Tris-Cl (buffering agent)
EDTA (metal chelator)
RNase A (degrades RNA)
6. Continue.
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Lyse cells with SDS/NaOH solution
Adding buffer P2 causes solution to become viscous
1. Sodium dodecylsulfate
Dissolves membranes
Binds to and denatures proteins
2. NaOH
Denatures DNA
Because plasmids are supercoiled, both DNA strands remain entangled after denaturation
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7. sodium dodecyl sulfate (SDS) potassium dodecyl sulfate (PDS)
(H2O sol. = 10%) (H2O sol. < 0.02%)
Continue
Neutralize NaOH with potassium acetate solution
Mixing with buffer N3 causes a fluffy white precipitate to form.
1. Potassium acetate / acetic acid solution
Neutralizes NaOH (renatures plasmid DNA)
Converts soluble SDS to insoluble PDS
2. Guanidine hydrochloride (GuCl)
Chaotropic salt; facilitates DNA binding to silica in
later steps
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8. Continue.
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Separate plasmid DNA from contaminants by centrifugation
Supernatant contains:
- Plasmid DNA
- Soluble cellular constituents
Pellet contains:
- PDS
- Lipids
- Proteins
- Chromosomal DNA
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Add cleared lysate to column and centrifuge
The high ionic strength and presence of chaotropic salt causes DNA to bind to the silica membrane, while other contaminants pass through the column
Centrifuge
Nucleic acids
Silica-gel membrane
Flow through
(discard)
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Wash the silica membrane to remove residual contaminants
Buffer PB contains isopropanol and GuCl
Centrifuge
PB buffer
Nucleic acids
Nucleic acids
PB + contaminants
Buffer PE contains ethanol and Tris-Cl
Centrifuge
PE buffer
Nucleic acids
Nucleic acids
PE + contaminants
(including residual GuCl)
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Elute purified DNA from the column
Buffer EB should be added directly to the membrane for optimal DNA recovery and to avoid possible EtOH contamination (from residual PE buffer)
EB is 10 mM Tris-Cl (pH 8.5). TE or dH2O may also be used.
Centrifuge
EB buffer
Nucleic acids
EB + DNA
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12. Continue.
PLASMID PREPARATION
1.5ml of bacterial culture was taken in centrifuge tube

Centrifuge of bacterial culture at 13,000 rpm 30 seconds

Collection of pellet in fresh eppendorfs tubes

Addition of 100l S1 buffer to the pellet

Addition of 200l S2 buffer and mixing of the sample by inverting 6-8 times

Addition of 150l of S3 buffer and mixing of the sample by inverting 6-8times

Addition of 450l of P1 buffer and mixing of the sample by inverting 6-8times

Centrifugation at 13,000 rpm 30 sec

Collection of supernatant in a fresh tube

Addition of 20l DBM into the supernatant and mixing the sample by inverting 6-8 times
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13. Continue...
Incubation at room temperature for 1 minute

Centrifugation at 13,000 rpm 30 sec

Removal of supernatant

Addition of 500l wash buffer to the pellet

Centrifugation at 13,000 rpm / 30 sec

Centrifugation until complete removal of wash buffer

Addition of 20lElution buffer to the pellet

Incubation at room temperature for 1 minute

Centrifugation at 10,000 rpm/30 second

Collection of Elutein a fresh tube

store in 20C in freeze
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14. Gel electrophoresis
Agarose gel electrophoresis is a widely used method that separates moleculesbased upon charge, size and shape. The purpose of the gel will be either to visualize the DNA,to quantify it or to isolate a particular band. Agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole.
DNA is visualized in the gel by addition of ethidium bromide , binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visibleorange light.
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15. PREP. Of 1% AGAROSE GEL
Take one 250 mg Agarose tablet in 25 ml of 1x TAE buffer. The tablet gets disintegrated with in 1 min. Mix the content and heat it in a microwave for 30 seconds.
Mix the content and allow the Agarose to cool to 50C and pour it in a plate that was sealed on either sides using a tape. After the gel is solidified, remove the tape and use it for electrophoresis of DNA samples.
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16. Continue.
Most Agarose gels:
1.1% gels are common for many applications.
2. 0.7%: good separation or resolution of large 510kb DNA fragments
3.2% good resolution for small 0.21kb fragments.
4.Up to 3% can be used for separating very tiny fragments but a
vertical polyacrylamide gel is more appropriate in this case.
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17. Continue
Buffer
The most common buffers for Agarose gel:
TAE: Tris acetate EDTA
TBE: Tris/Borate/EDTA
SB: Sodium borate.
TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.
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18. Continue..
An agarose gel is prepared by combining agarose powder and a buffer solution.
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Buffer
Flask for boiling
Agarose
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Combine the agarose powder and buffer solution.Use a flask that is several times larger than the volume of buffer.
Buffer solution
Agarose Powder
20. Melting the Agarose
Agarose is insoluble at room temperature
The agarose solution is boiled until clear
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21. Gel casting tray & combs
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22. Preparing the Casting Tray
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Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface.None of the gel combs should be touching the surface of the casting tray.
23. POURING THE GEL IN TRAY
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Allow the agarose solution to cool slightly (~60C) and then carefully pour the melted agarose solution into the casting tray.Avoid air bubbles.
24. Each of the gel combs should be submerged in the melted agarose solution.
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When cooled, the agarose polymerizes, forming a flexible gel.It should appear lighter in color when completely cooled (30-45 minutes).Carefully remove the combs and tape.
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Place the gel in the electrophoresis chamber.
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DNA
BUFFER
WELLS
ANODE (positive)
CATHODE
(Negative)
Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm.Make sure each well is filled with buffer.
28. Sample Preparation
6X Loading Buffer:
Bromophenol Blue (for color)
Glycerol (for weight)
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Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye).This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.
29. Loading the Gel
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Carefully place the pipette tip over a well and gently expel the sample.The sample should sink into the well.Be careful not to puncture the gel with the pipette tip.
30. Running the Gel
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Place the cover on the electrophoresis chamber, connecting the electrical leads.Connect the electrical leads to the power supply.Be sure the leads are attached correctly - DNA migrates toward the anode (red).When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.
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Cathode
(-)
DNA
(-)

wells
Bromophenol Blue
Anode
(+)
After the current is applied, make sure the Gel is running in the correct direction.Bromophenol blue will run in the same direction as the DNA.
32. Observe the gel under UV Light
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Assessing your plasmid preparation
1. Quantify abundance (A260) and purity (A260/A280)
2. Verify by restriction digestion
3. Run undigested plasmid to see if it is mostly
supercoiled
SUPERCOILED
DENATURED
33. END
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HIMANSHU CHAUDHRY

Himanshu chaudhry

Health & Medicine

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