2xzwei

So I just did this preliminary test in 12-well plates with few bacteria (3mL total volume which gave about 12ng/mL desired protein). I'm going to repeat this in larger scale (200mL culture) and hopefully the protein yield will be much higher...

The spin columns I found had only very low sample volumes (~100µL). Would it work to 1st precipitate the proteins with ammonium sulphate, dissolve the pellet in buffer and then go for spin columns?

Thanks for this suggestion! So how does this work - just put the sample on the column where all protein will be retained and then I can elute it? I've never done like this before.

Would it be reasonable to prior concentrate the proteins by ammonium sulphage / TCA precipitation?

I finally got it work. The Lysozyme I initially used was old. Ordered a new one and repeated the isolation process and it worked quite fine.

If you want to screen a large amount of colonies so instead of miniprep you can also do a colony PCR on a single colony. Just pick it from plate and use an aliquot for colony PCR with primers that are able to identify your plasmid uniquely. (I always resuspend the colony in 5µL dH2O, use 2.5µL for PCR reaction and with the other 2.5µL you can go for miniprep if it turns out that your colony is the one you need, store it at 4°C for 1-2 days or freeze).

PCR reaction follows the standard protocol:
5min heating to 95°C (where your bacteria gets lysed and DNA/plasmid is set free)

cycle 25-35 times with
30sec 95°C
30sec 56°C (dependent on primers)
30sec-2min 72°C (depends on amplicon size)

10minutes 72°C for final elongation, then do gel electrophoresis to check for correct amplicons. Colonys that show the right size can then be amplified and mini/maxi prepped.

If not the case - you can try to preliminary coat the plates with collagen type I. Depending on the culture flasks and cells you're using this might be beneficial.