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

http://hg.wustl.edu/hdk_lab_manual/plasmid/plsmid08.html

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 ul 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 ul per transformation but this can be adjusted to conserve cells. Do not use less than 60 ul 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 ul 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 ul 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 ul and 100 ul of each culture and plate on LBM Amp plates (if ampicillin is the selection) using a glass spreader. The 100 ul 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.
   

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