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Teknova,inc
主营:全数字超声诊断系统,全数字化彩色多普勒超声诊断系统
℡ 4000-520-616
℡ 4000-520-616
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cDNA文库的载体制备
1.pBlueScriptII的提取1.取1ul商品的pBlueScriptII,转化入大肠杆菌宿主菌中,取5ul转化产物均匀涂布在含AMP的LB平板上,37℃培养过夜。2.第二天取一只无菌的50ml离心管,加入10ml AMP抗性的LB液体培养基,挑单克隆于离心管中,37℃,250rpm,培养过夜。3.第三天取200µl小摇后的菌液接种于250ml 含AMP的LB液体培养基中,37℃,250rpm培养6 hr左右,使OD值达到0.6-0.8。4.将菌液移入250ml离心管中, 4℃,3000rpm,离心15min。取出离心管,菌团朝上倒掉上清,将离心管倒置于吸水纸上使上清充分滤干。(注意离心前需配平)5.加入10ml溶液Ⅰ(50mM Glucose , 25mM Tris-HCl,10mM EDTA, pH8.0),加入RNase至终浓度100µg/ml,晃动摇菌,使菌体充分悬浮,静置10min。6.按NaOH(0.4N):SDS(2%)--1:1的比例新鲜配制溶液Ⅱ,加入20ml溶液Ⅱ,静置3-5min。注: 静置时间勿超过5min,提前将溶液Ⅲ置于冰盒中。7.加入15ml冰浴的溶液Ⅲ,冰浴15-30min。8.4℃,5000rpm,离心15min。9.取上清于两个50ml离心管中,弃去原离心管中的沉淀。10.每管加入0.6倍体积的异丙醇,充分混匀,室温下放置10min。11. 20℃,12000g,离心20min回收质粒沉淀。12.弃上清,用70%的乙醇洗2次。13.弃上清,倒扣于吸水纸上,尽量空干液体。14.用3ml TE(pH8.0)溶解沉淀,移入1.5ml Eppendorf离心管中。15.电泳检查DNA质量并定量。(必要的话,可以用胶回收的方法先纯化一下质粒再进行双酶切。)2.pBlueScriptII的双酶切消化1.以如下体系进行EcoRI酶切:pBSK(+) X µl(6µg)ddH2O 174-X µl10×Buffer E 20 µl混匀,加入限制性内切酶:EcoRI (10U/ µl) 6 µl总体积为200 µl。2.轻弹管壁或用枪头轻轻吹打混匀,在离心机上甩一下。3.37℃,水浴1hr。4.加入200ul 1:1的酚/氯仿,混匀。4℃,13000rpm,离心15min。5.取上清,加入等体积的氯仿,4℃,13000rpm,离心10min。6.取上清,加入0.1倍体积的NaAC和2.5倍体积的无水乙醇,-20℃,沉淀30min。7. 4℃,13000rpm,离心10min,弃上清,取沉淀。8.加入200ul 70%的乙醇洗沉淀。9.4℃,13000rpm,离心10min,弃上清,取沉淀。10.自然风干沉淀,至无乙醇味,加入100ul ddH2O充分溶解沉淀。11.加入以下试剂进行XhoI酶切:ddH2O 74 µl10×Buffer D 20 µl混匀,加入限制性内切酶XhoI:XhoI (10U/ µl) 6 µl总体积为200 µl。12.轻弹管壁或用枪头轻轻吹打混匀,在离心机上甩一下13.37℃,水浴1.5hr。14.加入200ul 1:1的酚/氯仿,混匀。15.4℃,13000rpm,离心15min。16.取上清,加入等体积的氯仿,4℃,13000rpm,离心10min。17.取上清,加入0.1倍体积的NaAC和2.5倍体积的无水乙醇,-20℃,沉淀30min。18. 4℃,13000rpm,离心10min,弃上清,取沉淀。19.加入200ul 70%的乙醇洗沉淀。20.4℃,13000rpm,离心10min,弃上清,取沉淀。21.自然风干沉淀至无乙醇味,加入40ulddH2O充分溶解沉淀,得到双酶切载体。3.载体去磷酸化1.在40ul双酶切载体中加入以下试剂:6ul 10×buffer6ul CIAP(0.01U/ul)8ul ddH2O总体积60ul。2.轻弹管壁或用枪头轻轻吹打混匀,在离心机上甩一下。3.37℃,水浴1hr。4.70℃,15min,灭活酶。5.电泳分离,胶回收双酶切载体,定量。4.载体效率检测1.按以下所示作4个连接反应: DNA 连接酶 1 pBlueScriptII/E/X /CIAP - 检测酶切效率 2 pBlueScriptII/E/X /CIAP + 检测脱磷效率,载体自连效率 3 商品Vector加标准Insert + 对照 4 自制Vector加标准Insert + 检测载体效率 14℃连接过夜。2. 各取1ul连接产物作电转化(具体流程见电转化)3.计算克隆数,计算载体相对脱磷效率及连接效率。向左转|向右转
啊陌0187 2017-10-18
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上海起福成为Teknova 产品中国代理
2013-05-28
Teknova/BG-11 Media Agar Plates with Spectinomycin 10. 100mm, 20 plates, sterile. Cat.No. B4106/B410
2016-01-23
Teknova/Tyrosine Agar Plates. 100mm Plates, Sterile. Cat. No. T2530/T2530/
2016-02-16
Teknova/CM Glucose Plates minus Histidine. 150mm, 20 plates, sterile Cat.No. C3021/C3021/
2016-03-12
Teknova/10% Tween 20. 1000mL, Sterile. 6 Pack. Cat. No. T0124-06/T0124-06/
2016-03-18
Teknova/CM Glucose plates minus Ade, His, Leu and Trp plus 2mM 3AT. 100mm, 20 plates, sterile Cat.No
2016-04-09
Teknova/PCR Certified Water, RNase and DNase Tested. 1000mL, Sterile Cat. No. W3350/W3350/
2016-04-09
Teknova/MRS agar plates, 100mm, 20 plates, sterile Cat.No. M1350/M1350/
2016-05-10
Teknova/King's Medium A Plates Pseudomonas Agar F. 150mm, 20 plates, sterile. Cat.No. K0151/K0151/
2016-05-30
Teknova/M9 Minimal Salts Broth with Dropout minus Histidine. 500mL, sterile Cat.No. M8290/M8290/
2016-06-12
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Teknova/LB Broth with Animal-Free Soytone, 1 Kg. Cat. No. L8965/L8965/
Teknova/SOC Medium. 7mL. 10 pack. Sterile. Cat. No. S1657/S1657/
Teknova/Stain Extraction Buffer with DTT Separate Kit, 10-Pack 10X S0010 + 10X D0104 Cat.No. S0011/S0011/
Teknova/LB Lennox Broth. 454 g. Cat. No. L9305/L9305/
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Teknova特努瓦成立于1996年,是为了填补研究人员寻找预浇琼脂板或快速扭转定制产品的空白。Teknova established in 1996, was created to fill a void for researchers looking for pre-poured agar plates or custom products with a fast turnaround. Te
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Aperto Protein Extraction System
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Teknova/1X Blocking Buffer. 500mL, Sterile Cat. No. B1213/B1213/
$34.57
Teknova/C750501 13C Labeling Media, Auto-Inducing. 1L, Sterile. Cat.No. 3C7505/3C7505/
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Teknova/LB Broth with Animal-Free Soytone, 1 Kg. Cat. No. L8965/L8965/
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Teknova/SOC Medium. 7mL. 10 pack. Sterile. Cat. No. S1657/S1657/
$65.09
Teknova/Brain Heart Infusion (BHI) Agar Plates with 25 µg/ml Kanamycin. 100mm Plates, Sterile. 20 Pack Cat. No. B1097/B1097/
$42.26
Teknova/Stain Extraction Buffer with DTT Separate Kit, 10-Pack 10X S0010 + 10X D0104 Cat.No. S0011/S0011/
$625.11
Teknova/M9 Salts Media with 1% Glucose and 1 mM MgSo4. Sterile. Cat.No. 2M1990/2M1990/
$69.55
Teknova/1M Potassium Phosphate, Monobasic Solution. 1 L, sterile Cat.No. P0209/P0209/
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