Published: November 29th, 2019 Last Modified: December 13th, 2019
Getting the social media (“sosh meed”) train rolling to help grow the DIY Biology community. Follow me to get regular updates on when I post something new! And what better way to kick things off than to share a sneaky little protocol with you all?
Let’s say you’re doing a standard transformation to get a plasmid into BL21s for protein expression, or to get fresh colonies for a maxi prep. You’ve just had nice, fat burrito and the hour and a half protocol could be better spent on a nap. Skip all that! Heat shock your cells and plate just a whiff, you’ll get enough on your plate to do what you need to do. You only need one colony after all.
Competent cell protocol from Wendy Chao’s blog, thanks Wendy, wherever you are 🙂
*** Do not use this protocol for cloning reactions where you expect low efficiency, like a 4 fragment Gibson assembly/ InFusion or the like. Those experiments deserve the extra hour and a half finesse steps, you need every edge you can get! ***
Hi Alex,
Superb! Though did you try the same with some ligation / In-Fusion reaction? I know that SOC incubation is not required for ampicillin based plasmids. Does this work equally well with other antibiotic markers as well like CmR, SpecR or GentR?
On a second thougt, how much does the method used to prepare the chemically competent cells contribute. I generally use the CaCl2 method to prepare competent E. coli. Would this work as efficiently with my competent cells?
Thanks for the kind words and feedback Yogesh!
Yes, I should be more clear, I would not recommend this quick protocol for transformation of cloning reactions, there you usually need as high of a transformation efficiency as you can get. I’ll add that to the post. This is to save time when you’re transforming known plasmids of a decent purity which we tend to do pretty routinely. For example, if I wanted to re-transform a plasmid into BL21s for expression, this method would work, or if I wanted to get some fresh colonies for maxiprep, this would do the trick.
Should be pretty antibiotic agnostic, the efficiency of the competent cells will dictate how well this works.
On that note, next time you make up a batch of competent cells give Wendy’s Inouye variant a try, we used to do the traditional CaCl2 method and this is a significant upgrade, it gives you commercial levels of competence. Its quite a bit easier too, growing the cells at a lower temperature gives you more flexibility of when you harvest.
I will definitely try Mandy Chow’s modified Inoue. the buffer seems simple enough!
If I don’t want to use an aliquot of a high competence cell and have the time I normally scrape cells from a fresh plate, resuspend in 100uL (100mM CaCl2 + 10mM Tris pH8). Incubate 2h-4h on ice. Add DNA. Incubate for additional 30min. Heat shock 42°C for 90 seconds. Recover with SOC for 1hour. Useful when trying several expression strains.
Thanks for sharing this. Just tried it out, and in the final aliquoting step was carried out in a cold room. This protocol works well and now I have heaps of Chemical competent cells Mach1.
Ryan
That said, I’ll be comparing this to the RbCl2 method. My former colleague left me a protocol and apparently RbCl2 Transformation buffer (TB) is much more efficient than CaCl2 protocol.
I’ll get my summer student to give it a go in the new year, and if it proves to better then I’ll email you the TB recipe.
Ryan
I only use this buffer – 65 mM CaCl2, 20 mM TRIS 7.0, 15% glycerol. Grow, inoculate SOB, grow at 28 C (two days is too much) to OD 0.35-0.45, put on ice, pellet cells, resuspend in half volume (of the culture) of competent buffer, pellet cells, resuspend in 1/10 volume (of the culture) of competent buffer, incubate 30 min on ice, aliquot. For amp resistant cells I just add around 2-3 uL of plasmid, mix, incubate on ice for 10 mins and plate directly (like Zymo’s Z-competent protocol). It just works.
This is a bit more easier than modified Inoue but some chemicals (i.e. PIPES and MnCl2) is too much pricey here so this one costs a bit more less.
Whichever way you end up making your competent cells, it’s still cheaper than buying buying them a la cart from NEB or Thermo…5-40$ per 50uL reaction.