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
Verify selection for your plasmid/cosmid. You will need LBM plates with this antibiotic.
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.
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.
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.
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
Examine the plates and determine the efficiency of transformation.
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.coliDNA 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.
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.
Inoculate the plate with the transformed bacteria as before and incubate inverted at 37 degrees C for 16 hours.
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:
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.
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.
