IPTG and Xgal for bluewhite selectionIPTG and Xgal for bluewhite selection
IPTG and X-gal for blue/white selection
Stock Solutions
: isopropyl thiogalactoside, or isopropyl beta-D-thiogalactopyranoside. Sigma IPTG stock number I5502.
I use a 0.1 M solution. The formula weight is 238.3, so thi...
IPTG and Xgal for bluewhite selection
IPTG and X-gal for blue/white selection
Stock Solutions
: isopropyl thiogalactoside, or isopropyl beta-D-thiogalactopyranoside. Sigma IPTG 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.
: 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Sigma stock number X-gal 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., X-gal solutions
do not need to be sterilized.
Using IPTG and X-gal 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 X-gal in them, or incorporate the chemicals into top agar.
, Putting IPTG and X-gal on top of pre-made agar plates. Spread 40 ul of IPTG
and 40 ul of X-gal 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 X-gal into the plates before pouring. After
oauotclaving the media and cooling it to 65C or less, add IPTG to a final
concentration of 0.1 mM IPTG ( 1 ul IPTG stock solution per ml of media)
and X-gal to a final concentration of 40 ug/ml (2 ul of X-gal 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 X-gal 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
o50C. Add 10 ul IPTG stock and 40 ul of X-gal stock. Then add the bacteria
and phage mixture, mix quickly by rolling the tube between your palms, and
pour it onto the plate.
本文档为【IPTG and Xgal for bluewhite selection】，请使用软件OFFICE或WPS软件打开。作品中的文字与图均可以修改和编辑，
图片更改请在作品中右键图片并更换，文字修改请直接点击文字进行修改，也可以新增和删除文档中的内容。
该文档来自用户分享，如有侵权行为请发邮件ishare@vip.sina.com联系网站客服，我们会及时删除。
[版权声明] 本站所有资料为用户分享产生，若发现您的权利被侵害，请联系客服邮件isharekefu@iask.cn，我们尽快处理。
本作品所展示的图片、画像、字体、音乐的版权可能需版权方额外授权，请谨慎使用。
网站提供的党政主题相关内容(国旗、国徽、党徽..)目的在于配合国家政策宣传，仅限个人学习分享使用，禁止用于任何广告和商用目的。
浙公网安备 33021202002483