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I am an intern in biology institute. I have a 17.3kbp plasmid need to transform to Ecoli. But I have tried many time but have no or very few colony on LB plate.

I know large plasmid have less frequency in transformation, How can I improve chance of successful transformation?

Thanks everyone.

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  • $\begingroup$ Welcome to Biology.SE! It is expected that you will demonstrate that you have done research on your own before asking questions here — for example you could start with this: researchgate.net/post/… …. –––––––– Thank you for taking the tour — for details on how to ask good questions on this site please see: biology.stackexchange.com/help/how-to-ask. Thanks! 😊 $\endgroup$ – tyersome Jul 26 '19 at 4:30
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    $\begingroup$ Other than what tyersome has suggested, I want to add that questions that involve troubleshooting of lab experiments should include the detailed protocol used (or provide a link to a protocols article if it is exactly followed). $\endgroup$ – WYSIWYG Jul 26 '19 at 8:53
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The plasmid is quite big which might explain why it is difficult to transform bacteria (is it a lentiviral vector?). If you could give additional details you might have a chance to have improved answers to your question.

Hello, I suggest you follow this protocol:

At step 7 instead of this "Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. "

Do this " Add 250 μl LB or SOC media (without antibiotic) to the bacteria and grow in 30°C shaking incubator for 2 hours. Do not shake too hard, gentle motion might be fine (500 rpm).

At step 9 instead of this "Incubate plates at 37°C overnight."

Try to incubate at 30°C. I would even suggest to try to grow them at lower (25°C, or 16°C) temperature if you can prepare a second plate. It will take much longer than overnight to grow the cultures, probably more 48 hours (might be 72 hours at 16°C).

Hope that helps good luck!

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