Protocol: Transformation of Plasmids/Cosmids into E. coli

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Tony Rook
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Protocol: Transformation of Plasmids/Cosmids into E. coli

TITLE:
Transformation of Plasmids/Cosmids into E. coli

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.

METHODS:

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.
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
ul 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 ul
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 ul or several plates
of 200 ul for each ligation. Plate 100-200 ul 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 ul of a stock solution of X-gal (20
mg/ml) and 4 ul 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:

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)

SOC Media:
SOB + 20 mM Glucose

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

IPTG:
1. 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.

CITATIONS:

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

Matthew S. Holt http://humgen.wustl.edu/hdk_lab_manual/plasmid/plsmid08.html

Matthews Mokoba
Matthews  Mokoba's picture
Request for advice

I have followed your protocol To a pre-made LBM+ antibiotic plate added 40 ul of a stock solution of X-gal (20mg/ml) and 4 ul of a stock solution of ITPG (200 mg/ml).Spread the solutions over the entire surface of the plateusing a glass spreader.Incubated the plate at 37 degrees Cuntil all the fluid has been absorbed. Inoculated the plate with the transformed bacteria and incubate inverted at 37 degrees C now its 11hrs later but the plates only have blue colour on the agar surface without any colonies but on the LBM only the coloniesSo i want to ask what could be causing this?