Greetings everyone,
First of all, sorry for the late turnout but some of you have expressed interest in some of the DIY protocols I perform to run my lab on a lesser budget. I have opened this topic to share some of the protocols that I use routinely in the lab, especially for the reuse, recycling and in-lab generation of molecular biology reagents.
I will post here sporadically from time to time (sorry, poor time management skills and low Vitamin D for memory) so that you can all benefit and utilize them to make YOUR labs run cheaper.
Note - The protocols outlined here are not originals - they are mostly handpicked from the internet and various research papers. But, I have tried them numerous times and vouch for their applicability.
Note - Whatever chemical I use in the lab is the cheapest I can find. So they are not Sigma, Alfa Aesar, etc. For biologicals, I get my stuff from Bioshop Canada. For chemicals, I use BLDpharm. If I can find ever cheaper alternatives, I get OEM stuff (from local vendors that import and pack in their own containers).
I hope these stuff helps,
Sincerely,
Cihan
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- Express (Mostly) Anything, Anytime (E. coli) -
Expressing various proteins are especially important in running a molecular biology heavy lab cheaper, because enzymes cost much money. Having the ability to express and purify proteins in-lab is hence extremely important.
A regular E. coli based expression (provided that you have an appropriate recombinant plasmid) starts with transformation into BL21(DE3) (or variants), growing to OD600 ~0.6-1.0, and inducing with IPTG. For most proteins, it is advantageous to grow to early-mid log phase in 37 C so accumulate biomass faster, followed by induction at a lower T (16-25 C) to lower saturation of protein production apparatus and obtain more soluble protein. Even with Studier’s AI media most researchers still tend to do this - grow in 37 C for some time, then transfer to final T.
Recently, I have encountered some problems in my expression - even my plates were smelling acidic, I wasn’t getting any expression in cultures and the culture smell would burn my nostrils (the problem stemmed from having too much lactose in my cheap Typtone, so cells would start expressing protein prematurely due to the lack of glucose and get stressed, but does not matter). Plates were resolved by adding 1% glucose (to repress CAP and T7 polymerase production) but still, got problems in the culture. Then I tried this…
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Transform BL21(DE3) with plasmid and plate on LB + 1% Glucose plates
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Next day, scrape colonies with 1-2 mL of LB + 1% Glucose (pour the LB onto the plates, scrape the plate with a glass scraper to resuspend colonies, homogenize with a 200 uL pipette)
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Inoculate an AI-culture, 1:1000 dilution (e.g. 50 uL cells for 50 mL culture) (Note - At this point, depending on the number of your transformants you have a really thick inoculum, if it is on the ligther side, you can increase the amount of cells you can put into the AI culture) (Note - AI culture is Studier ZYM 5052 with Tryptone instead of NZ-Amine)
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Put the cultures in a RT shaker (30 C for thermophilic proteins)
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Collect after 30 hours (18 hours for thermophilic proteins)
To be continued…
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Thank you Cihan, that would be very very useful. My lab is full of devices that stay unused because of the lack of reagents.
Adrian