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Plasmid Isolation (Mini prep)
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Procedure:

 

  1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.
  2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.
  3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
  4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.
  5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents well by inverting the tube . Do not vortex! Store the tube in ice.
  6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube in ice for 3-5 minutes.
  7. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C in a microfuge. Collect the supernatant to a fresh tube.
  8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.
  9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.
  10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.
  11. Discard the supernatant by  aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.
  12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.
  13. Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not adhere tightly to the tube.
  14. Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).
  15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds and store the DNA  at -20°C.

 

Recipes for Buffers, Solutions and Media:

 

Alkaline Lysis Solution I :

 

50 mM glucose.
25 mM Tris-Cl (pH 8.0).
10 mM EDTA (pH 8.0).


Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by autoclaving and store at 4°C.
(For plasmid preparation.)

 

Alkaline Lysis Solution II:

 

0.2 N NaOH (freshly diluted from a 10 N stock).
1% (w/v) SDS.
Prepare Solution II fresh and use at room temperature.
(For plasmid preparation.)

 

Alkaline Lysis Solution III:

 

5 M potassium acetate, 60.0 ml.
Glacial acetic acid, 11.5 ml.
H2O, 28.5 ml.
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.
(For plasmid preparation.)

 

EDTA:

 

To prepare 0.5 M  EDTA  (pH 8.0):  Dissolve 186.1 g of disodium EDTA•2H2O in 800 ml of Distilled 2H2O. Stir well on a magnetic stirrer.  EDTA will not dissolve  into solution until the pH of the solution is reached to ~ 8.0 . So  the pH should adjust to 8.0 with NaOH (~ 20 g of NaOH pellets) and make up the final volume to 1000ml with distilled water. Prepare the  aliquots and sterilize by autoclaving.

 

Glycerol:

 

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a pre rinsed 0.22-μm filter. Store in 200-ml aliquots at 4°C.

 

LB Media:

Deionized H2O, to 950 ml.
Tryptone, 10 g.
Yeast extract, 5 g.
NaCl, 10 g.

To prepare LB (Luria-Bertani) medium, shake the ingredients , mentioned above with Distilled water until the solutes have dissolved. Adjust pH to 7.0 with 5 N NaOH and make up the final volume of the solution to 1 liter with deionized H2O. Then sterilize it for 20 minutes  by autoclaving  at 15 psi .

 

NaCl:

 

To prepare 5 M  NaCl : Dissolve 292 g of NaCl in 800 ml of  sterile H2O and the volume is make up to  to 1 liter with deionized H2O. Prepare the aliquots and sterilize it by autoclaving.

 

NaOH:

 

To 800 ml of H2O,  add 400g of NaOH pellets slowly, stirring continuously.  After  dissolving the pellets,completely, make up the final volume to 1 liter with sterile H2O. Store the solution at room temperature.

 

Potassium Acetate:

 

5 M potassium acetate, 60 ml.
Glacial acetic acid, 11.5 ml.
H2O, 28.5 ml.
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at room temperature

 

SDS:

 

Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of  SDS in 900 ml of H2O. Heat to a temperature of  68°C and stir with a magnetic stirrer to help dissolution.  Adjust the volume to 1 liter with  distilled H2O. Store at room temperature. Autoclaving not necessary.

 

STE:

 

10 mM Tris-Cl (pH 8.0).
0.1 M NaCl.
1 mM EDTA (pH 8.0).
Sterilize the solution by autoclaving and store at 4°C.

 

TE:

 

100 mM Tris-Cl (desired pH).
10 mM EDTA (pH 8.0).
(10x Tris EDTA) Sterilize the buffer by autoclaving and store at room temperature.

 

Tris-Cl:

 

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH  by adding concentrated HCl , to the desired value.The volume of the solution is make up to 1 liter with distilled H2O. Prepare the aliquots and sterilize by autoclaving. 

 

Procedure for Operating the Virtual Lab:

 

Check whether you have done all the steps listed below:

 

  • Prepare the culture containing the desired plasmid.
  • Incubate the culture for 24 hours at 37°C.
  • Take the culture from the incubator.
  • Transfer 1.5ml of the culture to a microfuge tube.
  • Centrifuge the tube for 30seconds at maximum speed (4°C).
  • Remove the supernatant.
  • Add 100μl alkaline lysis solution I.
  • Vortex the sample.
  • Add 200μl of alkaline lysis solution II.
  • Mix the sample by inverting the tube.
  • Store in ice for 1 minute.
  •  Add alkaline lysis solution III.
  • Mix the contents by inverting the tube.
  • Store in ice for 3-5 minutes.
  • Centrifuge the solution at maximum speed (4°C) for 5 minutes .
  • Collect  the supernatant to a fresh tube.
  • Precipitate the nucleic acid by adding 2 volumes of ethanol.
  • Mix by vortexing.
  • Stand the tubes for 2 minutes.
  • Centrifuge for 5 minutes.
  • Collect the precipitated DNA.
  • Discard the supernatant by aspiration.
  • Stand the tube as inverted to drain the fluid away.
  • Add 1ml 70% ethanol.
  • Mix by inverting.
  • Centrifuge the mixture for 2 minutes.
  • Discard the supernatant by aspiration.
  • Allow to dry for 3-5 minutes.
  • Add TE buffer with RNAse.
  • Mix by flickering.
  • Detect the plasmid by doing agarose gel electrophoresis.

 

 

Help:

 

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• on Ubuntu - https://help.ubuntu.com/stable/ubuntu-help/net-install-flash.html.en 

• on Fedora - https://docs.fedoraproject.org/en-US/quick-docs/using-adobe-flash/

 

 

 

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