2. plasmid extraction1) The plasmid used in this experiment is pET22b.2)Cultureconditions:LBliguidmedium(containingampicillin100 μg / ml) at 37°C 200 rpm shaker oscillation culturedovernight.3)Take3ml/twicebrothcentrifugation(thesameEPtuberepeated centrifugal twice), cells were collected4) plasmid extraction process : according the Instructons ofplasmidextractionkit(Axygen)
2. plasmid extraction 1）The plasmid used in this experiment is pET22b. 2）Culture conditions: LB liquid medium (containing ampicillin 100 μg / ml) at 37 ° C 200 rpm shaker oscillation cultured overnight. 3）Take 3ml/ twice broth centrifugation (the same EP tube repeated centrifugal twice), cells were collected 4) plasmid extraction process ：according the Instructons of plasmid extraction kit （Axygen ）
Note:1.After the first step of centrifugation, the culturesupernatant needs to be removed completely,2.The time of cell lysis (by adding solution 2) shouldnot be too long,3.After adding solution 3, the tube should be shakingupside down for sufficient mixing,4.Solution 1 contains RNase, and should be kept at 4 °C
Note: 1.After the first step of centrifugation, the culture supernatant needs to be removed completely; 2.The time of cell lysis (by adding solution 2) should not be too long; 3.After adding solution 3, the tube should be shaking upside down for sufficient mixing; 4.Solution 1 contains RNase, and should be kept at 4 ℃
·电泳检测所检测条件：1%琼脂糖凝胶电泳。配制方法：50mlTAE缓冲液+0.5g琼脂糖，微波炉加热沸腾至完全澄清加入5μlGOLDWAVE染色液体，插入小梳齿。点样：2.5μl样品+1μl6×或者10xLoadingBuffer点样。电泳时间：120V恒压30min分子量Marker1）DL2000，每次取5μl电泳时，750bp的DNA片段量约为150ng，显示亮带，其余条带的DNA量约为50ng。2）入DNAdigestDNA：在电泳前进行热处理（60℃5分钟）能使Marker的电泳图像变得更为清晰
• 电泳检测 检测条件：1%琼脂糖凝胶电泳。 配制方法：50ml TAE缓冲液+0.5g 琼脂糖，微波炉加热沸腾至完全澄清， 加入5µl GOLDWAVE染色液体，插入小梳齿。 点样：2.5 µl样品+1µl 6×或者10× Loading Buffer点样。 电泳时间：120V恒压 30 min 分子量Marker： 1）DL2000，每次取5 μl电泳时，750 bp的DNA片段量约为150 ng，显示 亮带，其余条带的DNA量约为50 ng。 2）λ DNA digest DNA ：在电泳前进行热处理（60℃, 5分钟），能使 Marker的电泳图像变得更为清晰
3. Electrophoresis analysis所1% agarose gel electrophoresis:1. 50ml TAE buffer +0.5 g agarose, microwave heating toboiling to fully clarified, adding 5ul GOLDWAVEstaining liquid, insert a small comb;2. 7.5 ul sample +1.5 μul 6 × or 10 × Loading Bufferspotting,3.120V constant voltage 30 min
3. Electrophoresis analysis 1% agarose gel electrophoresis: 1. 50ml TAE buffer +0.5 g agarose, microwave heating to boiling to fully clarified, adding 5μl GOLDWAVE staining liquid, insert a small comb; 2. 7.5 μl sample +1.5 μl 6 × or 10 × Loading Buffer spotting; 3. 120V constant voltage 30 min
Molecularweightofmarker具.DL2000:each5ul ofelectrophoresis,a750bpDNAfragment is about150ng,displaybrightband,theremainingbandsofDNAabout50ng.@.^DNAdigest DNA:Heat before electrophoresis (60°C, 5 minutes)enablingtheMarkerelectrophoretic imagebecomesmoreclearLamdaDNA/HindIIlD12000Kb-200023.139.446.56-10004.36-7502.02-5000-2500.56(0.13)-1000.625ugon0.8%TAEagarosegel
Molecular weight of marker: ①. DL2000: each 5 μl of electrophoresis, a 750 bp DNA fragment is about 150 ng, display bright band, the remaining bands of DNA about 50 ng. ②. λDNA digest DNA: Heat before electrophoresis (60℃, 5 minutes), enabling the Marker electrophoretic image becomes more clear. Lamda DNA/Hind III