亚洲国产精品二区久久,日本美女后入式午夜视频在线观看,国产污视频在线观看,欧美日韩国产精品中文字幕在线观看

上海士鋒生物科技有限公司
中級會員 | 第14年

13127537090

標(biāo)準(zhǔn)品
培養(yǎng)基
培養(yǎng)基原料 霍亂弧菌診斷血清 大腸艾希氏菌診斷血清 志賀氏菌屬診斷血清 沙門氏菌屬診斷血清 標(biāo)準(zhǔn)血清,診斷血清 抗生素藥敏紙片 微生物配套試劑 微生物生化管 管裝培養(yǎng)基 即用型液體培養(yǎng)基 一次性培養(yǎng)基平板 顯色培養(yǎng)基 臨床培養(yǎng)基 菌種保存培養(yǎng)基 四環(huán)素檢定、厭氧亞硫酸鹽還原桿菌檢測培養(yǎng)基 維生素檢測培養(yǎng)基 一次性衛(wèi)生用品衛(wèi)生檢測培養(yǎng)基 罐頭食品商業(yè)無菌檢測培養(yǎng)基 飲用水及水源檢測培養(yǎng)基 藥品、生物制品檢測培養(yǎng)基 化妝品檢測培養(yǎng)基 動物細胞培養(yǎng)基 啤酒檢驗培養(yǎng)基 軍團菌檢測培養(yǎng)基 支原體檢測培養(yǎng)基 小腸結(jié)腸炎耶爾森氏菌檢驗培養(yǎng)基 彎曲桿菌檢驗培養(yǎng)基 產(chǎn)氣莢膜梭菌、肉毒梭菌、厭氧菌檢驗培養(yǎng)基 阪崎腸桿菌檢驗培養(yǎng)基 溶血性鏈球菌檢測培養(yǎng)基 李斯特氏菌檢測培養(yǎng)基 弧菌檢測培養(yǎng)基 乳酸菌、雙歧桿菌檢測培養(yǎng)基 酵母、霉菌檢測培養(yǎng)基 檢測培養(yǎng)基 沙門氏菌、志賀氏菌檢驗培養(yǎng)基 大腸菌群、糞大腸菌群、大腸桿菌及腸桿菌科檢測培養(yǎng)基 細菌總數(shù)檢測,增菌培養(yǎng)基
抗體
生物試劑
細胞
菌株
血清
細胞分離試劑
試劑盒

Inoue法制備大腸桿菌超級感受態(tài)細胞方法

時間:2016-7-25閱讀:877
分享:

實驗步驟:

1、Inoculate from an overnight grown in LB.從培養(yǎng)過夜的LB平板上挑取單菌落 。

2、Grow in 250 ml "SOB" at 18℃ until OD600 = 0.6.(0.3)接種于250ml SOB,18度培養(yǎng)至OD=0.6。

3、On ICE for 10 minutes.菌液置冰上10分鐘。

4、Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4℃.4度2500g離心10分鐘。

5、Resuspend Cells gently in 80 ml of ice cold "TB".小心用80ml預(yù)冷TB重懸細胞。

6、On ice for 10 minutes.(30min)菌液置冰上10分鐘。

7、Spin at 2500 x g (5000 rpm in a Sorvall GSA,5500 rpm in a Sorvall SS-34,or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4℃.4度2500g離心10分鐘。

8、Resuspend cells gently in 20 ml of ice cold "TB".小心用20ml預(yù)冷TB重懸細胞。

9、Add DMSO to a final concentration of 7%.加入DMSO至終濃度7%。

10、PlAce on ice for 10 minutes.置冰上10分鐘。

11、AlIQuot into 1-2 ml and freeze in liquid nitrogen.分裝,液氮速凍 。

12、Store in liquid nitrogen.凍存。

SOB Medium and TB (Transformation Buffer)

SOB    2% (w/v) bacto tryptone    TB      
0.5% (w/v) yeast extract    10 mM Pipes
10 mM NaCl    55 mM MnCl2
2.5 mM KCl    15 mM CaCl2
10 mM MgCl2    250 mM KCl
10 mM MgSO4    在加入MnCl2之前先用5N KOH調(diào)pH值到6.7,adjust pH to 6.7 with 5N KOH prior to adding the MnCl2
thanks to Markus Schneemann for the tip!
pH 6.7 - 7.0 Note:
Competent cells are fragile (cell wall is thought to be weakened),therefore treat the cells gently when preparing these cells.Do not vortex or pippette up and down to resuspend the cells.Do not spin the cells at too great a speed (spinning down at 5000g will cause some cells to lyse).

Always keep the cells chilled when making competent cell.Do not let them warm up.

Freezing the cells appear to make cells more competent.

Some cell strains may work better than others (DHlpha works well in my hand).Note also that some cells (e.g.HB101)has greater recombination activity than others.

This method doesn't appear to work with BL21,so just grow the cells at 30 or 37 ℃ when making BL21 competent cells.However,it has been suggested that the efficiency of BL21 prepared using Inoue method may be improved by treating it with DTT before freezing (add to 3.5% v/v of a 2.2M DTT,10mM KAc pH6 solution and incubate 10 minutes on ice).

Heat shock time should be determined for different strains of cell.For DHlpha or JM109 use 30-45 sec.For BL21 use 120 sec.

Deactivate ligase prior to transformation.Ligase may reduce transformation efficiency.

Diluting the ligation mixture (~5x)can also increase transformation efficiecy by reducing the amount of reagents/contaminants that may affect transformation.Likewise it has been suggested that phenol/chloroform treatment may also increase efficiency,but it is probably too much trouble to bother trying.

The DNA added should not be more then 5% of the volume of competent cells used.The final DNA concentration should not exceed 5 ng/μl.

The method above should give a transformation efficiency of more than 108 cfu per μg of plasmid DNA (pUC or pBluescripts)with over 109 cfu possible.

Transformation efficiency has a roughly inverse relationship with the size of plasmids.Cells with deoR mutaion (e.g.DHlpha)can improved the transformation of large plasmid.Relaxed plasmids has ~3/4 of the transformation efficiency of supercoiled plasmid.

2 different plasmids can be transformed at the same time,or one after another.But they must be compatible (they cannot have the same replicon).

For routine transformation whereby efficiency of transformation is of no import,some of the steps may be shorten or omitted.For example,heat-shock step may be unnecessary and recovery incubation time at 37 ℃ can be reduced or omitted (but do note that this may depends on the antibiotic used for selection- for ampicillin-type antibiotics the incubation time is not really that important,therefore you can plate the cells straight after heat-shock if you wish.for other antibiotics,however,the incubation time may be essential).

Plating cells- dry 1.5% agar plates (exposed upside down)at 37 ℃ for 2-4 hours just before use,the plate should be able to soak up to 0.8-1 ml of media when plating.For blue-white selection,it is not necessary to make X-gal plate,just add X-gal+ IPTG direct to cells,mix and then plate.

Detergents may be detrimental to the transformability of the competent cells,therefore the glassware used for making competent cells should not be washed with detergents.Polycarbonate flask may also be used instead of glass flask.DMSO can dissolve polystyrene,therefore use polypropylene tubes.

When cloning difficult and less stable sequence (e.g palindrome,repeats,LTR sequences),it helps to grow transform cells at lower temperatures (25-30 ℃ or room temperature)in very rich media (e.g.Terrific Broth).Also terminate growth before reaching late stationary growth phase when grown in liquid media (i.e harvest cells at OD550 between 1 and 2).Use of stabilizing strain is also useful.

There are other methods of making competent cells- e.g.CaCl2 method,RbCl method which is more effective than CaCl2 method.Electroporation is supposed to give higher efficiency (up to 1010 transformants per μg plasmid claimed),but for the simple cloning that we do,its use is not warranted (and it's more expensive,more trouble than it's worth,etc.).

If a cooling shaker is not available- grow the cells at room temperature.More discussions on making competent cell as well as references can be found in TIBS articles "Preparing μltra-competent E.coli" and "Better competent cells"

It is also possible to transform cells straight from plate.It is convenient but you should expect low efficiency.See the following reference for more details (as well as more information on competent cells and other protocols):

會員登錄

×

請輸入賬號

請輸入密碼

=

請輸驗證碼

收藏該商鋪

X
該信息已收藏!
標(biāo)簽:
保存成功

(空格分隔,最多3個,單個標(biāo)簽最多10個字符)

常用:

提示

X
您的留言已提交成功!我們將在第一時間回復(fù)您~
撥打電話
在線留言
市长含着秘书的奶头| 国产区高清在线一区二区三区| 美女让我插她的骚逼| 澳门一区二区免费下线观看| 精品精品国产一区二区性色av| 亚洲精品自拍偷拍第一页| 欧美伦禁片在线播放| 好爽又高潮了毛片在线看| 黄色视频网在线观看| 欧美丰满大屁股女人的逼被操视频| 精品久久av免费一区二区三区| 欧美另类在线观看| 亚洲日韩不卡一区二区三区| 女人张开腿让男人捅个爽| 中文字幕你懂的av一区二区| 久久久一区二区三区日本| 国产精品免费99久久久| 大鸡巴操逼视频免费| 日韩 欧美 一区 二区三区| 束缚久久久久久免费高潮| 白丝袜子宫啊啊啊不要了| 亚洲精品自拍偷拍| 很黄很爽的免费视频大全| 大肌巴日小个子女人视频| 新视觉亚洲三区二区一区理伦| 2021国产一区二区岛国| 亚洲一区二区女同性恋免费看| 青青视频在线人视频在线| 3色w九九久久男人皇宫宕| 日本高清一区二区三区水蜜桃| 国产合区在线一区二区三区| 国产精品欧美久久久久久| 欧美亚洲另类天天综合网| 操逼动漫首页登录| 国产精品999午夜激情| 成人男女做爰免费视频网| 国产精品免费久久久久久| 美国大骚逼啊啊啊| 精品久久av免费一区二区三区| 日韩精品人妻一区二区免费| 操我骚逼抽插视频|