Plasmid Mini-Prep

PLASMID MINI-PREP

Last Update: December 2006

PREPARE SOLUTIONS

1. Glycerol mix (1 L):

Mix 25 g (liquid weight) of glycerol and add dH₂O to 1 L (Autoclave)

2. Potassium mix (1 L):

Mix 170 mL of 1M KH₂PO₄ (monobasic), 720 mL of 1M K₂HPO₄ (dibasic), and 110 mL of dH₂O (Autoclave)

3. TE_50/10 (100 mL):

Mix 5 mL of 1M Tris pH 7.5, 2 mL of 0.5M EDTA pH 8.0, and 93 mL of dH₂O

4. TE_10/1 (100 mL):

Mix 1.0 mL of 1M Tris pH 7.5, 200 mL of 0.5M EDTA pH 8.0, and 99 mL of dH₂O

5. 4M LiCl (100 mL):

Mix 17 g of LiCl with 100 mL of dH₂O (Autoclave)

6. LB media (1 L):

Mix 10 g of Bacto Tryptone, 10 g of NaCl, 24 g of Bacto Yeast Extratct, pH to 7.5 with NaOH, and dH₂O to 1 L. (Autoclave)

7. TB media (1 L):

Mix 12 g of Bacto Tryptone, 24 g of Bacto Yeast Extratct, and dH₂O to 900 mL. Autoclave (add 100 mL of Glycerol mix (after autoclave)

8. Lysis solution (30 mL):

Mix 1.5 mL of 20% SDS, 1.2 mL of 5M NaOH, and 27.3 mL of dH₂O

9. 3M KOAc pH 4.8 (100 mL)

Mix 29.4 g of potassium acetate, 40 mL of dH₂O, add Glacial acetic acid to pH 4.8, and dH₂O to 100 mL (Filter sterilize) (store at 4^oC)

10. 1M KH₂PO₄ (100 mL):

Mix 13.6 g of KH₂PO₄ (monobasic), and 100 mL dH₂O (Autoclave)

11. 1 M K₂HPO₄ (100 mL):

Mix 17.42 g of K₂HPO₄ (dibasic), and 100 mL dH₂O (Autoclave)

PROCEDURE - HOMEMADE PLASMID MINI-PREP

1. Grow a 2-4 mL culture ON from a single colony/stock in LB/TB media (3.6 mL TB + 0.4 mL Potassium mix + antibiotics)

2. Pellet 1.5-3 mL of culture in a 1.5 mL eppendorf tube and resuspend in 200 mL TE_50/10

3. Add 200 mL Lysis solution, mix (RT for ~5 minutes) and add 200 mL of 3 M KOAc and mix (keep on ice)

4. Centrifuge for 2-5 minute at 12,000 rpms and transfer supernatant to new tube

5. Centrifuge again to get rid of any left over precipitate and transfer to new tube

6. Add 0.8 Vols of 2-propanol and 0.1 Vols of 3M KOAc pH 4.8, mix by inversion, and centrifuge for 30 minutes at 12 krpms

7. Discard supernatant, wash once with 100% EtOH, spin again if pellet dislodges (only 5 minutes) and dry pellet in a SpeedVac (~5 minutes)

8. Resuspend pellet in 50 uL of TE _10/1

Optional: Add 1 mL of 21 mg/mL RNAse during digestion (or at the last step if DNA is not to be stored for long and checked right away) to get rid of RNA that may mask small fragments

PREPARE SOLUTIONS - WIZARD PLASMID MINI-PREP

1. Diatomaceous Earth (250 mg/mL):

Mix 25 g of Diatomaceous Earth with 500 mL of dH₂O. Let settle for 3 hrs. Aspirate water and leave 100 mL

2. Wash Solution:

Mix 12 mL of 5M NaCl, 1 mL of 1M Tris pH 7.5. 0.5 mL of 0.5M EDTA, 46.5 mL of dH₂O, and 70 mL of EtOH

3. Resin:

Mix 1 mL of Diatomaceous Earth, 25 mL of 4M Guanidine thiocyanate/HCl, 20mM Tris-HCl pH 7, and 20mM EDTA (Store in the dark)

PROCEDURE - WIZARD PLASMID MINI-PREP

1. Grow a 4 mL culture ON from a single colony/stock in TB media (3.6 mL TB + 0.4 mL Potassium mix + antibiotics)

2. Pellet 3 mL of culture in a 1.5 mL eppendorf tube and resuspend in 200 mL TE_50/10 (all on ice)

3. Add 200 mL Lysis solution, mix and add 200 mL of 3 M KOAc and mix

4. Centrifuge for 1 minute and transfer supernatant to new tube and mix with 1 mL of Resin

5. Put Magic Mini-column at the end of a 3 cc syringe, load the sample-resin mix and pass through column

6. Wash column with 2 mL Wash solutiona and spin column on an eppendorf tube for 20 secs

7. Elute DNA by adding 50 mL of TE_10/1 to column, let it sit for 1 minute at RT and spin on a new eppendorf tube for 30 secs

Note: if large plasmids/fragments, heat dH2O/ TE10/1 at 65^oC prior to elution

Note: columns can be regenerated by stirring in water, bringing the water to a boil, and then letting the water cool to room temperature while stirring

PROCEDURE - HOMEMADE WIZARD RESIN

HOMEMADE receipt for WIZARD resin to isolate plasmid and DNA fragments from low melting agarose gels

Diatomaceous earth (Sigma D5384) ( 25 g = 2500 mini preps)

Guanidine thiocyanate (Sigma G6639)

4 M guanidine tiocyanate, 50 mM Tris-HCl pH 7, 20 mM Na-EDTA

1. Transfer 25 g of diatomaceous earth to a 1 L beaker containing 500 ml dH₂O, vortex for 5 min at room temperature and let resin to settle for 2-3 hours. Aspirate supernatant and floating materials

2.Add autoclaved dH₂O to 100 mL and shake or vortex to resuspend the slurry (25 g of diatomaceous earth will swell to ~ 50 mL with water. When resuspended in a total volume of 100 ml, the concentration is 250 mg/mL). Transfer the well suspended slurry to 2 x 50 mL Falcone tubes and store in the dark at room temperature

3. Before use, mix the resin stock in dH₂O, and transfer 1 mL to 24 ml solution of 4M guanidine tiocyanate, 50 mM Tris-HCl pH 7, 20 mM Na-EDTA.

4. Store in the dark at room temperature

Optional: for larger amounts: Transfer the content of 1/2 falcone tubes (25 ml of 250 mg/ml suspension to a large bottle containing 600 mL 4M guanidine tiocyanate, 50 mM Tris-HCl pH 7, 20 mM Na-EDTA). Store in the dark at room temperature. Good for at least 1 year.

Use 1 mL of resin to isolate DNA from mini-prep (follow Promega Wizard protocol) and 1 ml of the same resin to isolate DNA from a low melting agarose gel