Transformation of Plasmids/Cosmids into E. Coli 质粒、粘粒转化大肠杆菌

Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine

Purpose:

To utilize competent E. coli bacteria to replicate a specific DNA fragment. Three methods are presented transformation of intact plasmids/cosmids ,transformation of ligation mixes and color selectability procedures.

Time required:

1.Transformation - 1 hour

2.Growth - approximately 16 hours for visible colonies

Special Reagents:

1.Competent Cells

2.SOB Media (needed for DH5-ALPHA cells only)

3.SOC Media (needed for DH5-ALPHA cells only)

4.X-gal (needed for color selection only)

5.dimethylformamide (needed for color selection only)

6.IPTG (needed for color selection only)

Preface:

We have four types of competent E. coli cells available for transformations: LM 1035, SURE, DH5-ALPHA, and XL1-BLUE. LM 1035 and DH5-ALPHA both work well for transforming intact plasmids/cosmids. They also work well when transforming ligation mixes but do not have color selectability. XL1-BLUE cells are used with inefficient ligations because non- recombinant colonies (bacteria with either uncut vector or re-circularized cut vector) turn a faint blue color on the plate while colonies harboring plasmids containing a cloned insert will remain white (see below for color selectability procedures). The XL1-BLUE strain grows more slowly than LM 1035 or DH5-ALPHA. DH5-ALPHA cells require SOB media and plates and LM 1035 and XL1-BLUE work well with LBM media and plates. SURE cells are best for use with cosmids because they are specially constructed to stop unwanted rearrangement events (often seen with large plasmids and cosmids).

Competent cells are stored in 300 μl aliquots in -80 degrees C and must remain on ice for the first half of these protocols. Once thawed the cells must be used or thrown away for they cannot be re-frozen. Protocols call for 100 μl per transformation but this can be adjusted to conserve cells. Do not use less than 60 μl per transformation.

When selecting for resistance to ampicillin transformed cells should be plated at low density (<10e4 colonies per 90 mm plate) and the plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase secreted into the medium from ampicillin-resistant transformants rapidly inactivates the antibiotic in regions surrounding the colonies. Thus plating at high density or incubating for long periods results in the appearance of ampicillin-resistant satellite colonies.

Procedures:

Transformation of intact plasmid/cosmid:

Day 1

1.Verify selection sequence for your plasmid/cosmid (usually ampicillin resistance but not always). You will need LBM plates with this antibiotic.

2.Place 5-10 ng of plasmid/cosmid DNA into a labeled sterile eppendorf tube. Have an empty tube labeled as a negative control. Competent cells cannot grow on antibiotic plates without a plasmid/cosmid carrying the resistance gene so the negative control plate should not grow colonies. If possible as a positive control use 1-5 ng of a plasmid (e. g. pBR322) that previously has transformed this batch of competent cells efficiently.

3.Place competent cells directly on ice after removing from -80 degrees C storage. As the cells thaw add 100 μl to each eppendorf tube. Flick the tubes to mix contents and place immediately on ice for 30 minutes.

4.Remove tubes from ice and incubate for 2 minutes in a 37 degrees C waterbath for a heat shock. The heat shock makes the cell close its "pores" and retain the plasmid/cosmid DNA. Add 900 μl of sterile 1X LBM (or SOC for DH5- ALPHA) to each tube and continue incubating at 37 degrees C for 30 minutes.

5.Because intact plasmids/cosmids transform efficiently (approximately 1x10e7 per ug of supercoiled DNA) you may wish to plate two dilutions of each to ensure isolated colonies. Take 10 μl and 100 μl of each culture and plate on LBM Amp plates (if ampicillin is the selection) using a glass spreader. The 100 μl plates should be quite dense. Flame the spreader between plates and allow to cool before using. Give the plates 5 minutes to absorb the inoculum then invert and incubate at 37 degrees C for 16 hours or until colonies are of the desired size. Store the remainder of the transformation mixture at 4 degrees C. (If necessary it can be reused for up to one week.)

Day 2

1.Examine the plates and determine the efficiency of transformation.

2.Pick isolated colonies to prepare miniprep DNA. Quantitate and verify plasmid/cosmid DNA with the appropriate restriction enzyme digests and 1kb ladder and lambda standards. If large amounts are needed follow the procedure for a large scale plasmid preparations when the plasmid/insert is verified.

Transformation of ligation mixes:

Day 1

1.Verify selection for your plasmid/cosmid. You will need LBM plates with this antibiotic.

2.Pipet 1/2 of the prepared ligation mixture and two controls (see ligation protocols) into eppendorf tubes. You may wish to plate competent cells alone on a selection plate as a negative control. The remainder of the ligation mix should be stored at -20 degrees C and used as a back-up if necessary.

3.Place the competent cells directly on ice after removing from the -80 degrees C freezer. As the cells thaw add 100 μl to each eppendorf tube. Flick the tubes to mix and place immediately on ice for 30 minutes.

4.Remove tubes from ice and incubate for two minutes at 37 degrees C in a waterbath for a heat shock. Add 900 μl of sterile 1X LBM (or SOC for DH5-Alpha) to each tube and continue incubating at 37 degrees C for 30 minutes.

5.The efficiency of tranformation depends on the ligation mix so you may wish to plate 100-200 μl or several plates of 200 μl for each ligation. Plate 100-200 μl of each on LBM antibiotic (SOB antibiotic for DH5- Alpha) using a glass spreader. This amount is usually sufficient to obtain the isolated colony. Flame the spreader between plates and cool before each use. After inoculum has been absorbed (5 minutes) invert plates and incubate at 37 degrees C for 16 hours or until colonies are of the desired size. Store remaining transformation culture at 4 degrees C. This can be reused if necessary for up to one week.

Day 2

1.Examine the plates and determine the efficiency of transformation.

2.Pick isolated colonies to prepare miniprep DNA. Quantitate and verify plasmid/cosmid DNA with the appropriate restriction enzyme digests and 1kb ladder and lambda standards. If large amounts are needed follow the procedure for a large scale plasmid preparations when the plasmid/insert is verified.

Color selection:

Many vectors carry a short segment of E.coli DNA that contains the regulatory sequences and the coding information for the first 146 amino acids of the beta-galactosidase gene. Although neither the host-encoded nor the plasmid-encoded fragments are themselves active they can associate to form an enzymatically active protein. The Lac bacteria that result from beta-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galacoside (X-gal). However insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is not capable of beta-complementation. Bacteria carrying recombinant plasmids therefore form white colonies.

X-gal is very expensive but the cost can be minimized by spreading concentrated solution of the sugar on the surface of the plate rather than incorporating it in the media.

1.To a pre-made LBM antibiotic plate (or SOB antibiotic for DH5- Alpha) add 40 μl of a stock solution of X-gal (20 mg/ml) and 4 μl of a stock solution of Isopropylthio-Beta-D-Galactoside (ITPG) (200 mg/ml). Spread the solutions over the entire surface of the plate using a glass spreader. Incubate the plate at 37 degrees C until all the fluid has been absorbed. Because of the low volatility of dimethylformamide this can take up to 4 hours.

2.Inoculate the plate with the transformed bacteria as before and incubate inverted at 37 degrees C for 16 hours.

3.Place plates at 4 degrees C for several hours to enhance blue color.

Solutions:

1.SOB Media:

2% Bactotryptone

0.5% Yeast extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl₂

10 mM MgSO₄

1.5% Agar (for plates)

2.SOC Media:

SOB   20 mM Glucose

3.X-gal:

Dissolve 100 mg of X-gal in 5 ml of dimethylformamide in a sterile polypropylene tube. Aliquot 1 ml into eppendorf tubes wrapped in foil (to prevent damage by light) and store at -20 degrees C.  It is not necessary to filter sterilize X-gal solutions.

4.IPTG:

Dissolve 2 g of IPTG in 8 ml  of dH₂O in a sterile polypropylene

tube.  Adjust the volume to 10 ml  with dH₂O and filter through a 0.22

micron syringe filter into 1 ml aliquots and store at -20 degrees C.

References:

Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press p.1.74.