Plasmid DNA preparation by the toothpick method in small quantities
Plasmid DNA preparation by the toothpick method in small quantities
Colonies picked with a toothpick from agar medium can be used directly to prepare plasmid DNA. the resulting closed-loop DNA often has too many impurities to be used as a substrate for most restriction enzymes. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Plasmid DNA preparation experiment by toothpick method in small quantity
Principle
Colonies picked with a toothpick from agar medium can be used directly to prepare plasmid DNA. the resulting closed-loop DNA tends to have too many impurities to be used as a substrate for most restriction enzymes.
Materials and Instruments
Antibiotics Bromophenol blue solution EDTA Ethidium bromide NSS solution KCl Agarose gel Move I. Materials For more product details, please visit Aladdin Scientific website.
LB, YT, or SOB thermal cycler Wooden toothpick
1. Buffers and solutions
(1) Antibiotics for screening plasmids
(2) Bromophenol blue solution (0.4%, m/V) or cresol red solution (10 mmol/L)
(3) EDTA ( 0.5 mol/L, pH 8.0 )
(4) Ethidium bromide (10 mg/ml ) or SYBR Gold
(5) KCl ( 4 mol/L)
(6) NSS solution: 0.2 mol/L NaOH, 0.5% SDS, 20% sucrose.
2. Gel
(1) Agarose gel (0.7%, 5 mm thick), prepared and electrophoresed with Tris-acetate or Tris-acetate buffer without ethidium bromide.
When there is only a small difference in plasmid size, Tris-borate electrophoresis buffer (TAE) should be used for agarose gel electrophoresis because of its greater ability to solubilize superhelical DNA of different sizes. Otherwise, Tris-borate-EDTA ( TBE) buffer should be used because of its greater buffering capacity.
(2) Agarose gel
3. Culture medium
(1) LB, YT or SOB (high nutrient broth medium for E.coli)
(2) LB, YT or SOB agar plates with appropriate antibiotics
4. Specialized equipment
(1) Thermal cycler
(2) Water bath set to 70°C
(3) Wooden toothpick
II. Methods
1. Cell preparation
(1) Cultivate colonies transformed with the appropriate plasmid on enriched agar medium (LB, YT, or SOB) containing appropriate antibiotics until they reach 2~3 mm in diameter (for most strains, incubate at 37℃ for about 18~24 h).
(2) Using a sterile toothpick or disposable ring, transfer a small piece of the bacterial colony to a strip or a small piece of agar master plate containing the appropriate antibiotic, and transfer the remainder of the colony to a numbered microcentrifuge tube containing 50 μl of sterile 10 mmol/L EDTA (pH 8.0).
In addition to the solid colonies, several "empty" colonies (plasmid vectors without exogenous genes) should be transferred. These samples can be used as a reference for molecular quality standards in gel electrophoresis.
(3) Repeat step 2 until the desired number of colonies are harvested.
(4) When all colonies have been replicated and transferred, incubate the master plate at 37°C for a few hours and store at 4°C until gel electrophoresis results are obtained. Then colonies with plasmids of the expected size were harvested from the mother plate.
(5) While incubating the master plate, the bacterial suspension was processed as follows; 20 μl of freshly configured NSS solution was sequentially added to each microcentrifuge tube. Cap the tubes and mix by shaking for 30 s. The tubes were then capped with a cap.
(6) Transfer all centrifuge tubes to a hot water bath at 70°C for 5 min, then cool at room temperature.
(7) Add 1.5 μl of 4 mol/L KCl solution to each tube and mix for 30 s. Shake well.
(8) Place the tubes on ice for 5 min.
(9) Centrifuge the tubes at maximum speed for 3 min at 4°C to remove bacterial debris.
2. Gel electrophoresis analysis of plasmids
(1) Transfer the supernatant sequentially into clean centrifuge tubes. If the sample is to be analyzed by agarose gel electrophoresis only, add 0.4% bromophenol blue solution to each tube; if the sample is to be analyzed by agarose electrophoresis as well as PCR, add 2 μl of 10 mmol/L Methylphenidate to the sample. Add 50 μl of supernatant to the wells (5 mm long, 2.5 mm wide) of a 0.7% agarose gel (5 mm thick).
The agarose gel does not contain ethidium bromide and the buffer used does not contain ethidium bromide because the mobility of the superhelical DNA reflects its molecular mass more accurately than when the dye is inserted. This makes it easier to distinguish between plasmids of different sizes.
(2) When bromophenol blue has migrated to 2/3 or 3/4 of the gel, or cresol red has migrated to half of the gel, the gel is immersed in ethidium bromide solution (0.5 μg/ml aqueous solution) at room temperature for 30~45 min. the gel is observed under a UV light and photographed.
(3) If cresol red is used in (1), it is preferable to analyze the supernatant by PCR (using the residual liquid of each sample as a template).
Cresol red (Merk Index) is orange to amber in acidic solutions, yellow at pH 7.2, and red in alkaline solutions. Unlike bromophenol blue and xylene blue, cresol red does not inhibit the thermal stability of DNA polymerase (Taq polymerase, derived from thermophilic archaea) (Hoppe et al. 1992). Therefore, DNA solutions containing cresol red can be used in most amplification reactions. The migration of this dye in a 2% agarose gel lies between xylene blue and bromophenol blue and is approximately 300 bp in size ( Hoppe et al. 1992 ). On agarose gel electrophoresis photographs, cresol red does not produce shadows like ethidium bromide staining.
(4) Inoculate colonies on the mother plate that may contain recombinants into 2 ml of liquid medium (LB, YT, or SOB ) containing the appropriate antibiotic for small-scale culture.
It is not necessary to wait until the colonies on the mother plate grow to a large size. Even a thin layer of bacteria is sufficient for inoculation.
(5) Use this small-scale culture to prepare the recombinant sub-plasmid to be tested in small quantities. Analyze the plasmid DNA by enzymatic digestion and agarose electrophoresis to confirm that the size and structure are as expected.