I understand that you have ligated the insert, isolated in step 2, into pET-TEV28. I would recommend to transform your ligation from step 4 into XL-1 Blue , do a few small DNA preps and characterize DNA by restriction enzyme digestion.

The gel will show what is going on. Take the perfect plasmid to transform BL-21. Go ahead with protein induction. No further PCR is necessary.

so, I should transform my ligation into XL-1Blue, perform a mini prep and then run a gel with the DNA I got, right?

Thank you in advance!

Hello Raianna,

First, some bacteria strains have naturally developped resistance to some antibiotics. Maybe the colonies you see on the petri dish are just resistant bacteria with no plasmid? Try plating 50ul of your BL-21 alone on a petri dish.

Second, did you do a construct integrity control ? You should perform a plasmid extraction from your transformed BL-21 and run it on a gel electrophoresis to see what you got inside them. (optional: as Uwe Borgmeyer said you could do a restriction analysis to verify that it is indeed the right plasmid)

Finally, before plating on a kanamycine plate, I would recommend you to culture the transformed BL-21 (BL-21 + pET-TEV28) in 1ml LB medium (at 30deg C and 240rpm shaking) with 15ug/ml kanamycine for 1,5 hours then plating 50uL of the 1ml culture on a Kanamycine 50ug/ml LB-agar petri dish.

Hi Raianna,

Yes, just digest a few 100 ng of your mini preps, with Nhe I and Xho I or other suitable enzymes of the MCS.

Don't hesitate to share the hopefully positive outcome with us. If everything is as predicted, you have enough DNA to transform your BL21 whenever you have to produce your protein.

But Amer is right, you always have to exclude a contamination.

Best,

Uwe

Raianna Fantin ,

Transforming a ligation to a pET vector directly into BL21(DE3) is poor practice. If for some reason you have some amount of basal expression (for instance due to lactose contamination of tryptone; see e.g. PMID 15915565, 9524234, 21523618) or the encoded protein is slightly toxic, your chances of retrieving a recombinant clone decrease. Also, BL21 is a recA+ endA+ strain, so the quality and yield of plasmid preps will suffer.

Best to err on the safe side and use a strain devoid of T7 RNA polymerase. XL1-Blue, as suggested by Uwe Borgmeyer , is a great choice.

Hope it helps!

Alejandro Martin Uwe Borgmeyer

Thanks for all recommendations!

I found out that my antibiotic was actually not working well on petri dishes (it was working perfectly in culture medium, so we rule out the possibility at first). After I changed my antibiotic, it worked! Thanks anyway for the amazing insights! Best regards!

Raianna Fantin

Hello back,

Yes indeed this happens frequently O2 oxidation sensible antibiotics.

Thanks for sharing your output with us and best of luck for your subsequent work.

Regards