What protocol do you follow to get the best results? Since I am new to this technique, in this experiment I have some difficulties with the results.
Thanks in advance .
IPTG and Xgal for blue/white selection
Stock Solutions
IPTG : isopropyl thiogalactoside, or isopropyl beta-D-thiogalactopyranoside. Sigma stock number I5502.
I use a 0.1 M solution. The formula weight is 238.3, so this is 0.238 g in 10 ml of water. Sterilize by filtration, then store in the freezer.
Xgal : 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Sigma stock number B4252.
I use a 20 mg/ml solution. It must be dissolved in DMSO (dimethyl sulfoxide) or dimethyl formamide, not water! It must be wrapped in foil to protect it from the light, and stored in the freezer. According to Maniatis et al., Xgal solutions do not need to be sterilized.
Using IPTG and Xgal for blue/white selection on Petri plates
There are three basic methods: spread the chemicals on top of the plates before you use them, pour the plates with IPTG and Xgal in them, or incorporate the chemicals into top agar.
Putting IPTG and Xgal on top of pre-made agar plates. Spread 40 ul of IPTG and 40 ul of Xgal on top of the plate with a hockey stick spreader. Then, let the plates dry before you use them. This should take 30 minutes or so if the plate is dry (i.e. a day or two old), but up to several hours for freshly made plates. I definitely prefer this method for bacteria.
Incorporating IPTG and Xgal into the plates before pouring. After auotclaving the media and cooling it to 65oC or less, add IPTG to a final concentration of 0.1 mM IPTG ( 1 ul IPTG stock solution per ml of media) and Xgal to a final concentration of 40 ug/ml (2 ul of Xgal stock solution per ml of media). Also be sure to add the selection drug at this time: usually ampicillin to a final concentration of 100 ug/ml.
Putting IPTG and Xgal into top agar. This method is generally used for bacteriophage, but also works for bacterial colonies. Use 3 ml of 0.7% agar (or agarose if you want DNA that can be cut with restriction enzymes) kept at 50oC. Add 10 ul IPTG stock and 40 ul of Xgal stock. Then add the bacteria and phage mixture, mix quickly by rolling the tube between your palms, and pour it onto the plate.
Consider also that, if your cloned fragment is rather short, also the light-blue colonies can be the right ones if there are no white colonies.
besides blue white colony screening also check by plating on antibiotic containing plates if your desired target is inserted in any antibiotic site(amp/tet) on plasmid or genome.
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