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Friday, February 3, 2012

Plasmid Extraction / plasmid prep protocol & principle


Plasmids are double stranded, circular, extra chromosomal, self replicating DNA molecules which carry additional genes which helps the bacteria to survive in extreme conditions of stress.

Many methods have been developed to purify plasmids, these methods involves these steps
  • Growth of Bacterial Culture.
  • Harvesting and Lysis of Bacterial Cells.
  • Purification of Plasmid DNA.

Bacterial Plasmid - Isolation / Extraction
Bacterial Plasmid - Image Source

Growth of Bacterial Culture:

overnight culture of bacterial cells having plasmid can be used for plasmid extraction/ plasmid prep. The volume of bacterial culture used depends on the amount of plasmid required. if the plasmid has any markers like antibiotic genes, suitable antibiotic can be used which ensures only plasmid containing bacteria grows in it, why it is because bacterial cells tend to loose plasmid sometimes.

Harvesting and Lysis of Bacterial Cells:

Once the culture attains particular OD, depends on the amount you are going to start with, bacterial cells can be harvested by centrifugation. After centrifugation, Cells will settle at the bottom which can be collected and lysed for plasmid extraction.

Purification of Plasmid DNA:


Plasmid can be purified using silica columns.

Reagents Required:

Solution I

(Tris pH 8.0, EDTA- Ethylene diamine tetra acetic acid and Glucose)

RNase

Lysozyme- optional

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

Prepare Solution I from standards stocks of 100ml and autoclave for 15mins.

Solution II

0.2N Sodium hydroxide (Freshly diluted from 10N NoaH Stock)
1% SDS- Sodium dodecyl sulphate

(Alkaline pH 12.0)

Solution III
5M Potassium acetate – 60ml
Glacial Acetic acid – 11.5ml
Water – 28.5ml
(Acidic pH 5.4)

Plasmid Extraction / plasmid prep Principle


Plasmid prep process


Image Source: Favorgen Biotech Corp



Solution I
Solution I has EDTA in it which chelates magnesium and calcium ions, there by destabilizing the membrane. Glucose helps to maintain the osmolarity. Lysozyme which can be used to lyse the membrane.

Solution II
NaOH which is highly alkaline, SDS which creates pores on the cell wall and NaOH helps in loosening the cell wall, non-supercoiled DNA gets denatured at higher pH (due to NaOH), hydrogen bonding in DNA molecule is broken down and the two strands separates, but supercoiled plasmid DNA will be intact.

Solution III
Sodium or potassium acetate , when the acidic solution is added the separated strands reaggregates and forms a tangled mass, on centrifugation this insoluble network gets pelleted and the plasmid DNA will be left out in supernatant.

Protocol: Plasmid Extraction from E.coli

1. Transfer 1.5ml of bacterial culture to a micro centrifuge tube.

2. Pellet cells by centrifuging at 12,000 rpm for 2 minutes

3. CAREFULLY remove the supernatant.

4. Add 250 ul of Solution I /RNAase.

5. Resuspend the pellet by vortexing briefly or by pipetting up and down.

6. Add 250 ul of Solution II

7. Mix GENTLY by inverting and rotating the tube several times. DO NOT vortex!!!

8. Leave at room temperature for 5 minutes but NOT MORE than 5 minutes.

9. Add 350 ul of Solution III

10. Mix by inverting the tube 6-8 times

11. Centrifuge at 12,000 rpm for 5 min

12. While your tubes are spinning place a Spin column in a 2-ml collection tube.

13. After centrifugation, CAREFULLY transfer the supernatant to the column. DO NOT disturb the pellet.

14. Centrifuge the column and collection tube at 12,000 rpm for 1 min.

15. Discard the flow through collected in the collection tube.

16. Replace the column in the collection tube

17. Add 250 ul of 70% ethanol into the column and centrifuge for 1 minute at 12,000 rpm, discard the flow through, reapeat step 17

18. Place the column in a sterile 1.5ml tube and leave in room temperature for 2 mins.

19. Add 50ul of deionised water or TE buffer into the column.

20. Centrifuge at 12,000 rpm for 1 min.

21. Discard the column. Plasmid DNA is in the deionised water or TE buffer will be collected in the 1.5ml tube.

22. Place the tube containing plasmid DNA on ice for further use or store at –20oC.

DNA concentration can be checked using nanodrop spectrophotometer, and the analysis of the extracted plamsid can be done by running on an agarose gel.

There are various plasmid preparation / plasmid prep kits which can be used to extract the DNA like QIAGEN Plasmid Extraction kit, Promega Plasmid Extraction kit, Plamid isolation kit from GenScript , etc

Plasmid Extraction Kit
Qiagen Plasmid Extraction Kit - Image Source: Qiagen

References

J. Sambrook, Molecular Cloning 3rd Edition

TA Brown, Gene Cloning & DNA Analysis

QIAGEN Plasmid Extraction Technical Notes

Internet Sources