您的位置 首页 DNA/RNA技术

电转化的一些技巧与经验

发布: 2018-06-06 699阅读 201评论

原文链接：https://international.neb.com/tools-and-resources/usage-guidelines/electroporation-tips

Electroporation Tip

1. Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice.

2. Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes.

3. Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency.

4. 对于连接反应，可以用乙醇沉淀DNA，而后用水溶解以去除盐离子。Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than 10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns are recommended for clean up of ligation reactions.

5. Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap.

6. Arcing may occur due to high concentration of salts or air bubbles.

7. It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.

8. 预热琼脂板可以提高转化效率。Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.

9. 对于未使用完的感受态细胞，可以重新冷冻。Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.

10. 对于Bio-rad Micropulser, EC1 的出厂设置条件是1.8 kV，通常用于0.1 cm电转杯；EC2的设置是2.5 kV，常用于0.2cm 的电转杯；EC3的设置是3.0 kV。

11. 对于E. coli, 最佳的电转化条件是使用0.2 cm电转杯，40 uL 细胞，2.5 kV，时间常数(time constant)约5 ms

12. 对于酵母S. cerevisiae， 最佳的电转化条件是0.2 cm电转杯，40 uL细胞，1.5 kV电压，时间常数是5 ms

 

1.16K赞
标签:



相关文章

发表回复

您的电子邮箱地址不会被公开。 必填项已用*标注

评论列表(201)

  5. 📯 Reminder: + 1.8276 BTC. Next =>> https://telegra.ph/Go-to-your-personal-cabinet-08-25?hs=4e53c53dc13f499e21c8e9be30c4f694& 📯说道：

    vi2j8o

    回复
  9. 🗒 You have received a notification # 747. Open > https://telegra.ph/Go-to-your-personal-cabinet-08-25?hs=4e53c53dc13f499e21c8e9be30c4f694& 🗒说道：

    5745ud

    回复

  21. “What ya lifting now?” His dad asks as he fidgets on the commode seat. He can feel his hard-on growing in his pants.

    回复

  30. “That your jockstrap on the floor next to you, son?” His dad asks.

    回复

  34. “Stoke it, boy! Stroke it! Stroke that beautiful cock!” His father demands as his own cock draws on the wet interior of the glass enclosure shower. “Pound it harder, boy! Pound it harder!” “But you didn’t though, did you, son?”

    回复

  37. “Was it because of that?” His dad asks as he points a finger towards him. And his midsection. “What happened at today’s practice today that was so different from any other day, son?” His dad asks.

    回复

  38. Thanks, Valuable stuff!
    online casino platforms for real money [url=https://usacasinomaster.com/#]top rated online casinos[/url] bandar betting sbobet casino online

    回复

  39. Wow quite a lot of useful facts.
    buy online casino [url=https://luckyusaplay.com/#]online casino free bonus[/url] best online live casino uk

    回复

  40. The one shower after his workout and practice had been a good start. But the walk home had tired him. But the massaging jets of water caressing across his muscular frame takes the weariness from his bones from this stressful day. porno dla dzieci “FUCK! FUCK!” He says, as he loosens the tightened grip on his fleshy-red-tool, while it throbs with its life-giving blood coursing through its many vein-filled region.

    回复

  41. Nicely put. Regards.
    online casino gambling poker slot [url=https://luckyusaplay.com/#]monkeytilt best online casino[/url] online casinos zonder nederlandse vergunning

    回复

  46. Really tons of wonderful data!
    huff and puff casino game online [url=https://usacasinomaster.com/#]usa casino online[/url] casino online wunderino

    回复

  48. You said it adequately..
    online casino ghana [url=https://luckyusaplay.com/#]brand new online casinos usa no deposit bonus codes[/url] calientecasimo online gratis casino

    回复

  51. Cheers! I value it!
    atlantic club casino online [url=https://usaplayerscasino.com/#]live casino online[/url] williams interactive online casinos

    回复

  53. Really all kinds of great info.
    online casino belgiГ« [url=https://usacasinomaster.com/#]social casinos online[/url] online casino europe

    回复

  54. You actually explained it exceptionally well!
    dragon star online casino [url=https://luckyusaplay.com/#]online casino bonus no[/url] new online crypto casinos

    回复

  58. You’ve made your position very clearly..
    casino online bono por registro [url=https://usaplayerscasino.com/#]online casino bonuses[/url] top online casino in canada

    回复

  60. Seriously plenty of fantastic advice!
    echt geld online casinos [url=https://usagamblinghub.com/#]casino gambling online[/url] 2018 online real money casinos usa

    回复

  62. You actually suggested that effectively!
    colosseum casino online [url=https://usacasinomaster.com/#]casino online casino[/url] agen judi casino online

    回复

  64. Excellent write ups. Appreciate it.
    casino online betfair [url=https://luckyusaplay.com/#]best paying online casino[/url] casino solverde online

    回复

  66. Really a lot of superb info!
    nicaraguan online casino [url=https://uscasinoguides.com/#]casino onlines[/url] 666 casino online review

    回复

  69. Wow loads of awesome tips.
    how to bet online rivers casino [url=https://usacasinomaster.com/#]online games casino[/url] apply online casino

    回复

  71. Appreciate it, Ample tips!
    best online casinos in new jersey [url=https://luckyusaplay.com/#]online casino deposit bonus[/url] casino spiele online

    回复

  74. Very good info. Many thanks!
    colombia online casinos [url=https://usaplayerscasino.com/#]online casino free play[/url] casino online snai

    回复

  78. Thanks a lot! I value it.
    online casino schnell geld verdienen [url=https://luckyusaplay.com/#]online casino bonuses[/url] casino online tiradas gratis

    回复

  80. With thanks. An abundance of stuff.
    most popular online casino slots [url=https://uscasinoguides.com/#]best casino online[/url] vegas casino online bonus

    回复

  82. With thanks. I like this!
    wizard online casino [url=https://usagamblinghub.com/#]online usa casinos[/url] online casino with neosurf

    回复

  85. Beneficial data. Regards.
    no deposit online casino bonus codes usa [url=https://uscasinoguides.com/#]online casino free bonus no deposit[/url] free casino slot machine games online

    回复

  87. Superb information, Many thanks!
    online casino real money usa no download [url=https://usaplayerscasino.com/#]best usa online casinos[/url] hollywood casino online slots facebook

    回复

  89. Nicely put, Thanks a lot.
    online casino faq [url=https://usagamblinghub.com/#]best online casinos that payout[/url] raja casino online

    回复

  91. You actually explained this superbly.
    best online casinos ny [url=https://usacasinomaster.com/#]most trusted online casinos for usa players[/url] online casino genting malaysia

    回复

  93. Thank you! Ample content.
    online casino usa real money fast payout [url=https://luckyusaplay.com/#]new online casino no deposit bonus usa[/url] live online casinos

    回复

  97. Incredible all kinds of valuable advice.
    play casino online uk [url=https://luckyusaplay.com/#]casino online games[/url] skrill online casinos

    回复

  140. Great read! Your breakdown of the topic is commendable. For further reading, here’s a useful resource: READ MORE. Let’s discuss!

    回复
联系我们

联系我们

(44)07934433023

在线咨询: QQ交谈

邮箱: info@bioengx.org

关注微信
微信扫一扫关注我们

微信扫一扫关注我们

关注微博
返回顶部