Team:METU Turkey/Results Discussion/Plasmid purification

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Revision as of 21:28, 27 October 2010

MAIN STEPS/TIME TABLE

- Incubation [12-14 h]
- Plasmid purification [30-45 min]

MATERIALS

- FERMANTAS GeneJET™ Plasmid Miniprep Kit (#K0502 or #K0503)
- Centrifuge (M03)
- Nanodrop
- Spectrophotometer
- Incubator with shaker (M15)
- Heat block
- P1000, P200 and P10 micropipettes

- sterile dH2O
- LB broth with appropriate antibiotic
- 15 mL falcon
- 1.5 mL epp

CHECK-LIST PROCEDURE

Important
- Check availability of LB broth with appropriate antibiotic
- Check presence of sterile dH2O
- Check incubator with shaker and centrifuge to be worked properly
- Look plasmid. Is it high-copy or low-copy?
- Prepare 5 mL of E.coli culture in LB media for purification of high-copy plasmids.
- For low-copy plasmids use up to 10 mL of culture.
- All purification protocol should be carried out at room temperature.
- All cenrifugations should be carried out @ 10000-14000 rpm. (depending on rotor type) (We used @ 13000 rpm.)
- Check the Lysis Solution and the Neutralization Solution for salt precipitation use.
- Redissolve any precipitate by warming the solution at 37 C, then cool back down to 25 C before use. Do not shake the Lysis Solution too vigorously.
- Pick a single colony
- Inoculate in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented with the appropriate selection antibiotic.
- Incubate @ 225 rpm not longer than 12-14 h at 37 C .
- Spin @ 4000 rpm for 5 min at 25 C .
- Discard the supernatant and keep pellet.
- Resuspended the pellet with 250 ul Resuspension solution. (Bacteria should be resuspended completely by vortexing until no cell clumps remain)
- Transfer the cell suspension to epp.
- Add 250 uL Lysis Solution
- Mix throughly by inverting the tube 4-6 times until the solution becomes slightly clear.
- Do not vortex!
- Do not incubate for more than 5 min. (To avoid denaturation of supercoiled plasmid DNA!)
- Add 350 uL Neutralization solution
- Mix immediately and throughly by inverting the tube 4-6 times. (The neutralized bacterial lysate is cloudy and viscous) ("throughly" to avoid localized precipitation of bacterial cell debris)
- Spin @ 13000 rpm for 5 min to precipitate cell debris and chromasal DNA.
- Transfer the supernatant to spin column (600 uL civari). (Avoid disturbing or transferring the white precipitate)
- Spin @ 13000 rpm for 1 min.
- Discard the flow-through.
- Place the column back into same collection tube.
- Add 500 uL Wash solution to spin column.
- Spin @ 13000 for 1 min
- Discard the flow-through.
- Repeat this step with using 500 uL Wash solution.
- Discard the flow-through
- Spin @ 13000 for an additional 1 min to remove residual Wash solution. (This step is essential to avoid residual ethanol in plasmid preps)
- Transfer spin column into a fresh 1.5 mL epp.
- Add 50 uL Elution buffer into center of the spin column membrane to elute the plasmid DNA
- Do not contact the membrane with pipette tip!
- Incubate for 2 min at 25 C. >> To increase yield incubation is done for 2 min at heat block at 42 C
- Spin @ 13000 for 2 min.
- Discard the column and store the purified plasmid DNA at -20 C.

- OPTIONAL= additional elution step with elution buffer or water. This step increases the yield by 10-20 %.
- NOTE: For elution of plasmids ≥ 20 kb, prewarm Elution buffer to 70 C before applying.

Quality Check / Validation
(Concentration / DNA purity / Target purity check)

- Alpha UV Spec
- Restriction Enzyme Digestion (single digestion)
- Agarose gel electrophoresis

SOLUTIONS

TROUBLESHOOTING

Low yield of plasmid DNA
- Bacterial culture too old

 
- Inoculate a fresh batch of antibiotic-containing growth medium with a freshly-isolated single bacterial colony from an overnight plate.
 
- Cultivate the cells for no more than 16 h at 37°C while shaking in LB media. 
 
- Reduce the cultivation time to less than 12 h when using rich media like TB.

- Incomplete lysis of bacterial cells

 
- It is essential that the cell pellet is completely resuspended in the Resuspension Solution prior to lysis. 
No cell clumps should be visible before the addition of the Lysis Solution.
 
- Check the Lysis Solution for salt precipitation before each use.
 
- Redissolve any precipitate by warming the solution to 37°C, then mix well before use.
 
- Cell cultivation in LB media is recommended. 
 
- Reduce culture volume when using a rich cultivation media like TB.

- Inefficient elution of DNA

 
- The Elution Buffer must be dispensed to the center of the membrane for efficient elution.

Contaminated DNA preparation
- Residual ethanol

 
- Ensure that step 9 of the protocol is performed. 

- RNA in the eluate

 
- Ensure that RNase A is added to the Resuspension Solution before the first use.

- Genomic DNA in the eluate

 
- To avoid shearing and liberation of genomic DNA, do not vortex or shake the cells during lysis and neutralization(steps 2 and 3), mix by gentle inversion of the tube.
 
- Do not lyse the cells (step 2) for more than 5 min.
 
- Do not cultivate cells longer than 16 h in LB media or 12 h in TB media.

- Additional band of denatured plasmid

 
- Denatured plasmid DNA migrates ahead of supercoiled DNA. It is not suitable for following enzymatic manipulations such as restriction digestion.
 
- To avoid denaturation, do not lyse the cells (step 2) for more than 5 min.