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All protocols that I found about Lambda red recombination are using Electroporation as a method to introduce (inject) the homologous DNA (usually a PCR products or a linear dsDNA) to the E.coli cell. Can I use chemical method (CalCl2 + PEG) and heat shock method instead ?

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  • $\begingroup$ Rubidium chloride method and the Inoue's method of making chemical competent cells give higher efficiency than the CaCl₂ method. $\endgroup$ – WYSIWYG Dec 10 '18 at 10:10
  • $\begingroup$ @WYSIWYG thank you for this info. But "Rubidium" sounds very strange to me. if it not popular, as I might think it will expensive than CaCl2 . $\endgroup$ – joe Dec 10 '18 at 11:23
  • $\begingroup$ It is certainly more expensive than calcium chloride. Rubidium an alkali metal and you can check out the protocol for RbCl competent cell preparation, here. It also uses other salts. These salts may be slightly more expensive than CaCl₂ (because these metals are rarer) but would still be cheaper than buying an electroporator and the cuvettes. $\endgroup$ – WYSIWYG Dec 10 '18 at 11:28
  • $\begingroup$ @WYSIWYG Thank you alot. But any evidence that RbCl will work with lambda red recombination? If electroporate is just firing a high voltage between 2 electrode, I'm planning to purchase a high voltage generator module on Ebay, edit it a bit and put it directly to a plastic tube. Will be much cheaper? $\endgroup$ – joe Dec 10 '18 at 13:07
  • $\begingroup$ You can make a DIY electroporator but I don't think an Ebay product will work that well (you'll just end up wasting more time). You need to generate a pulse and you need to have proper electrodes. I have not worked with lambda red. This site says that recombination rate is less than 1:10000. You can get efficiency of ~10⁷/µg with RbCl method which means you should get ~10³ colonies. You can use a selectable marker to select the positive cells. $\endgroup$ – WYSIWYG Dec 10 '18 at 13:17
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Since Lambda red recombination is a rare event you want to have to have a good transformation efficiency, thus electroporation would be your best choice. Also since the expression of the recombination system usually needs growing bacteria at 42º you will probably inhibit the expression of the recombination machinery with the chemical method.

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  • $\begingroup$ Thank you for answer. Because electroporator is so expensive so I'm finding a more cheaper protocol. $\endgroup$ – joe Dec 10 '18 at 6:47

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