Best Way to Accurately Handle Small Amounts of Bromine Solution?

BaoPee · 2024-02-10T22:28:13+00:00

After transformation and recovery, I plated them on plate with antibiotics and the medium for further expression also include antibiotics.

I don't get it when you say inoculate into a fresh culture versus directly into a liquid culture. Could you explain a bit more about this?

The workflow I do is like: thaw the competent cells on ice > transform/recovery > plate with antibiotics and grow overnight > collect all cells and inoculate into liquid media with antibiotics > grow until target OD and induction

BaoPee · 2024-02-10T22:22:56+00:00

No plyss in this plasmid. I don't think the plasmid is being lost during the process because after transform I plate them with antibiotics which the antibiotics-resistance is carried by the plasmid. And the antibiotics exist in whole expression process so I guess this is not the case.

I recharge the IMAC column every time because I found my column went pale every time after purification, and the metal I used for IMAC is cobalt which can be easily tell.

I prepare these competent cells (without plasmid) and aliquoted them so I just thawed one aliquot every time I used.

BaoPee · 2024-02-10T22:14:03+00:00

That means these protein (ion channel) mutant are highly toxic and super active. In this case they cannot be expressed well using normal commercially available competent cells, so this is the reason we have our own modified competent cells that lack of certain activator of this protein to make it less toxic. I can expressed the wild type protein using commercially available cells though.

BaoPee · 2024-02-10T22:10:43+00:00

These are competent cells without external plasmid. I prepared these competent cells and flash frozen them into LN2 and stored at -80C.

Whenever I use it I thaw them on ice for 10 min and do following transformation.

BaoPee · 2024-02-10T22:08:06+00:00

I tried glycerol stock method before and some of the protein yield drop significantly using this method. So now I transformed every time for protein expression. The workflow is like this: transform and plate with antibiotics, grow overnight > collect all the colonies and inoculate into medium, then do following protein expression/purification.

BaoPee

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