Paul.xu at 23:34, 10 November 2011

2011-11-10T23:34:25Z

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'''7 Steps Protocol''' 

'''S I ''' 50 mM Tris-HCl, 10 mM EDTA, pH 8.0; 

'''S II''' 

0.2 M NaOH, 1% SDS 

 '''S III '''

2.0 M acetic acid, 0.6 M potassium acetate, 0.4 M manganese chloride, 0.4 M calcium chloride 

 

1. Re-suspension of the host cells in S I of 50 mM Tris-HCl, 10 mM EDTA, pH 8.0; 2. Lysis of the cells by adding equal volume of S II of 0.2 M NaOH, 1% SDS; 3. Neutralizing the lysate with equal volume of S III of 2.0 M acetic acid, 0.6 M potassium acetate, 0.4 M manganese chloride, 0.4 M calcium chloride; 4. Obtaining clarified lysate by centrifugation or filtration; 5. Precipitate plasmid DNA from the clarified lysate by adding PEG 8000 to a final concentration of 4%; 6. Collect the plasmid DNA precipitation by centrifugation or filtration; 7. (optional) Dissolve the plasmid DNA in water, and precipitate the plasmid DNA again by adding calcium chloride to a final and concentration of 40 mM and PEG 8000 to a final concentration of 4%. Collect the final plasmid DNA precipitation by centrifugation or filtration. 

 

'''Mini-Prep 2ml scale:''' 

1. Inoculate 2 ml culture medium with a single E. coli colony containing appropriate plasmids. Grow overnight at 37° C. with vigorous shaking; 

 

2. Transfer 1.5 ml of the bacteria culture into a microfuge tube. Centrifuge at 8,100 g for 1 min. Discard the supernatant; 

3. Add 150 µl of S I [50 mM Tris-HCl, 10 mM EDTA, pH 8.0] into the tubes. Vortex to re-suspend the pellet; 

 

4. Add 150 µl of S II [0.2M NaOH and 1% SDS] to the bacterial suspension. Close the tube and mix the contents by inverting the tube several times to lyse the bacteria;  

5. Add 150 µl of S III [2.0 M HAc, 0.6M KAc, 0.4 M MnCl 2 , 0.4 M CaCl 2 ] to the tube. Close the cap and mix the content by inverting the tube several times. A white precipitation should form; 

 

6. Centrifuge the bacterial lysate in a microfuge at full speed for 2 min. Transfer the supernatant to a new tube;  

7. Add 50 µl of 40% PEG 8000 to the clarified lysate. Close the tube and invert a few times to mix the contents;  

8. Centrifuge the tube in a microfuge at full speed for 2 min. Pellet is not visible after the centrifuge. Discard the supernatant;  

9. Add 500 µl of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Spin for 2 min and discard the liquid; 

 

10. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette;  

11. Add 50-100 µl of water to dissolve the plasmid DNA. 

'''Midi-Prep'''

1. Inoculate 5 to 30 ml culture medium with a single E. coli colony containing appropriate plasmids. Grow overnight at 37° C. with vigorous shaking; 2. Spin down the bacteria at 3000 ~ 6000 rpm for 5 min. Discard the supernatant; 3. Add 600 &micro;l of S I [50 mM Tris-HCl, 10 mM EDTA, pH 8.0] into the tubes. Vortex to re-suspend the pellet; 4. Transfer the bacteria into a 2 ml microfuge tube. Add 600 &micro;l of S II [0.2M NaOH and 1% SDS]. Close the tube and mix the contents by inverting the tube until the bacteria are completely lysed; 5. Add 600 &micro;l of S III [2.0M HAc, 0.6M KAc, 0.4M MnCl 2 , 0.4 M CaCl 2 ] to the tube. Close the cap and mix the content by inverting the tube several times. A white precipitation should form; 6. Centrifuge the bacterial lysate in a microfuge at full speed for 10 min. Transfer the supernatant to a new 2 ml microfuge tube; 7. Add 200 &micro;l of 40% PEG 8000 to the clarified lysate. Close the tube and invert a few times to mix the contents; 8. Centrifuge the tube in a microfuge at full speed for 5 min. A translucent pellet can be seen. Discard the supernatant; 9. In an optional step, add 900 &micro;l of water to the tube. Vortex to dissolve the plasmid DNA. Add 100 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to the tube. Close the tube and invert several times to mix the content. Centrifuge the tube in a microfuge for 2 minutes to pellet the plasmid DNA; 10. Add 500 &micro;l of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Disregard the liquid; 11. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 12. Add 100-200 microliter of water to dissolve the plasmid DNA.

 '''Maxi-Prep'''

1. Grow 100 ml E. coli culture overnight at 37° C. with vigorous shaking; 2. Transfer the culture to a centrifuge tube and spin down the bacteria at 4,500 g for 5 min. Discard the supernatant; 3. Re-suspend the bacteria in 6 ml of S I [50 mM Tris-HCl, 10 mM EDTA, pH 8.0]; 4. Lyse the bacteria by adding 6 ml of S II [0.2M NaOH and 1% SDS]; 5. Neutralize the lysate with 6 ml of S III [2.0M HAc, 0.6M KAc, 0.4M MnCl 2 , 0.4 M CaCl 2 ]; 6. Centrifuge the lysate at 13,000 g for 10 min (see alternative protocol below at 24). Transfer the supernatant to a new tube; 7. Add 2 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents. Centrifuge the tube at 13,000 g for 10 min. Discard the supernatant; 8. Dissolve the pellet in 0.9 ml of water and transfer to a 1.7 ml microfuge tube. Add 100 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to the tube. Close the tube and invert several times to mix the content. Centrifuge the tube in a microfuge for 5 minutes to pellet the plasmid DNA; 9. Add 1.5 ml of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Disregard the liquid; 10. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 11. Add 200-500 microliter of water to dissolve the plasmid DNA; 12. Alternatively, the bacterial lysate can be processed by filtration: transfer the lysate to a syringe that has a 5 micron syringe filter attached. Wait for 2 minutes for the white aggregate flow to the top. Push the lysate through the syringe filter into a new tube; 13. Add 2 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents; 14. Transfer the lysate to a syringe that has a 0.1 micron syringe filter attached. Push the mixture through the syringe filter to capture the plasmid DNA precipitation on the filter; 15. Remove the syringe plunger. Add 1.0 ml of water to the syringe, and push through with the plunger into a 1.7 ml microfuge tube. Repeat the procedure again, but with 0.45 ml of water, and collect the elution into the same tube; 16. Add 135 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to the tube. Close the tube and invert several times to mix the content. Centrifuge the tube in a microfuge for 5 minutes to pellet the plasmid DNA; 17. Add 1.5 ml of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Disregard the liquid; 18. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 19. Add 200-500 microliter of water to dissolve the plasmid DNA.

 '''Mega-Prep'''

1. Grow 500 ml E. coli culture overnight at 37° C. with vigorous shaking; 2. Spin down the bacteria at 4,500 g for 5 min. Discard the supernatant; 3. Re-suspend the bacteria in 24 ml of S I [50 mM Tris-HCl, 10 mM EDTA, pH 8.0]; 4. Lyse the bacteria by adding 24 ml of S II [0.2M NaOH and 1% SDS]; 5. Neutralize the lysate with 24 ml of S III [2.0M HAc, 0.6M KAc, 0.4M MnCl 2 , 0.4 M CaCl 2 ]; 6. Centrifuge the lysate at 13,000 g for 10 min (see alternative protocol below at 24). Transfer the supernatant to a new tube; 7. Add 8 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents. Centrifuge the tube at 13,000 g for 10 min. Discard the supernatant; 8. Dissolve the pellet in 1.8 ml of water and transfer to a 2.0 ml microfuge tube. Add 200 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to the tube. Close the tube and invert several times to mix the content. Centrifuge the tube in a microfuge for 5 minutes to pellet the plasmid DNA; 9. Add 2.0 ml of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Disregard the liquid; 10. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 11. Add 0.5 to 1.0 ml microliter of water to dissolve the plasmid DNA; 12. Alternatively, the bacterial lysate can be processed by filtration: transfer the lysate to a syringe that has a 5 micron syringe filter attached. Wait for 2 minutes for the white aggregate flow to the top. Push the lysate through the syringe filter into a new tube; 13. Add 8 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents; 14. Transfer the lysate to a syringe that has a 0.1 micron syringe filter attached. Push the mixture through the syringe filter to capture the plasmid DNA precipitation on the filter; 15. Remove the syringe plunger. Add 0.9 ml of water to the syringe, and push through with the plunger into a 1.7 ml microfuge tube. Repeat the procedure again and collect the elution into the same tube; 16. Add 200 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to the tube. Close the tube and invert several times to mix the content. Centrifuge the tube in a microfuge for 5 minutes to pellet the plasmid DNA; 17. Add 2.0 ml of 15% isopropanol to the tube. Close the tube and invert the tube a few times. Disregard the liquid; 18. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 19. Add 1 ml of water to dissolve the plasmid DNA.

 '''Giga-Prep'''

1. Grow 2.5 liters of E. coli culture overnight at 37° C. with vigorous shaking; 2. Transfer the culture to a centrifuge tube and spin down the bacteria at 4,500 g for 5 min. Discard the supernatant; 3. Re-suspend the bacteria in 120 ml of S I [50 mM Tris-HCl, 10 mM EDTA, pH 8.0]; 4. Lyse the bacteria by adding 120 ml of S II [0.2M NaOH and 1% SDS]; 5. Neutralize the lysate with 120 ml of S III [2.0M HAc, 0.6M KAc, 0.4M MnCl 2 , 0.4 M CaCl 2 ]; 6. Centrifuge the lysate at 13,000 g for 10 min (see alternative protocol below at 24). Transfer the supernatant to a new tube; 7. Add 40 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents. Centrifuge the tube at 13,000 g for 10 min. Discard the supernatant; 8. Dissolve the pellet in 3.6 ml of water and transfer to two 2 ml microfuge tube. Add 200 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to each tube. Close the tubes and invert several times to mix the content. Centrifuge the tubes in a microfuge for 5 minutes to pellet the plasmid DNA; 9. Add 2.0 ml of 15% isopropanol to the tubes. Close the tubes and invert the tubes a few times. Disregard the liquid; 10. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 11. Add 1 ml of water to each tube to dissolve the plasmid DNA and combine the contents of the two tubes; 12. Alternatively, the bacterial lysate can be processed by filtration: transfer the lysate to a syringe that has a 5 micron syringe filter attached. Wait for 2 minutes for the white aggregate flow to the top. Push the lysate through the syringe filter into a new tube; 13. Add 40 ml of 40% PEG 8000 to the clarified lysate. Close the cap and mix the contents; 14. Transfer the lysate to a syringe that has a 0.1 micron syringe filter attached. Push the mixture through the syringe filter to capture the plasmid DNA precipitation on the filter; 15. Remove the syringe plunger. Add 1.8 ml of water to the syringe, and push through with the plunger into a 2.0 ml microfuge tube. Repeat the procedure again and collect the elution into another tube; 16. Add 200 &micro;l of 0.4 M calcium chloride in 40% PEG 8000 to each tube. Close the tubes and invert several times to mix the content. Centrifuge the tubes in a microfuge for 5 minutes to pellet the plasmid DNA; 17. Add 2 ml of 15% isopropanol to the tube. Close the tube and invert a few times. Disregard the liquid; 18. Briefly spin the tube in a microfuge to collect all liquid to the bottom. Aspirate all the liquid using a pipette; 19. Add 1 ml of water to each tube to dissolve the plasmid DNA. Combine the contents of the two tubes 

[[Category:Protocols]]

Fast-MiniPrep - Revision history

Paul.xu at 23:34, 10 November 2011