How can I check if the kanamycin is working?

I have a problem with kanamycin - why the kanamycin doesn't affect?

Hi everyone, I need to expression of GFP with pET28 plasmid in E. coli BL21(DE3) plysE. Transformed cells have kanamycin and chloramphenicol resistance. Normally, only transformed cells should...

22 February 2021 4,032 4 View

What is the optimal propagation regime for pET-1M(KanR) and pMCSG7(AmpR) plasmids in DH5alpha E.coli?

Dear all, I am trying to harvest the low-expression plasmids in the mentioned E.coli strain. I constantly gel very low concentrations (30-50 ng/uL while the concentration is usually around 100...

08 February 2021 4,255 4 View

Why is there no fluorescence if GFP-expressing vector is present and sequence is correct?

We recently constructed a plasmid vector with a high-copy number ori, kanamycin resistance, and eGFP under control of the lac promoter but lacking the lac operator. However, the cells are not...

19 January 2021 7,834 2 View

Can I use G418 for selection of HEK 293 cell lines that already have a plasmid with kan and Hygro integrated?

I am doing a kill curve in HEK 293 cells with G418(0-1000ug/mL) and they don't die. They were previously transfected and selected with hygromycin and are integrated with a plasmid has also...

05 December 2020 3,232 3 View

Best way for knockout of gene in Bacillus genome?

Hi everyone! I would like to knockout a gene in the genome of Bacillus. To do that I tried to make a construct that has 1) a backbone, 2) a kanamycin resistance cassette, flanked by 3) the...

24 November 2020 489 1 View

How to grow yeast BY4743 with XX::MX4 - which medium components are needed?

I want to grow yeast (BY4743) with a certain knock out (deletion), the deletion cassette is KANMX4. I assume I have to use 200mcg/ml geneticin. But can anyone explain step by step how i have to...

19 November 2020 3,434 3 View

PMaxGFP bacterial transformation failure?

I bought a Lonza nucleofector transfection kit a few months ago, which has a pMaxGFP plasmid in it. Then we want to use this plasmid as the reference gene expression in some other experiments and...

14 September 2020 2,273 6 View

What is a good ligation strategy with compatible ends EcoRI and MfeI in the vector DNA and the insert?

I used to do this kind of thing and now have forgotten strategy please help and be specific. The vector is now linear having been digested with EcoRI and MfeI, then treated with CIAP, to prevent...

23 May 2020 2,377 1 View

Why I got relaxed plasmid from E. coli maxiprep?

Hi. I have faced a problem I never seen before. I have Origene pCMV-XL6 Entry plasmid without any ORF - just empty plasmid. It has Kanamycin resistance so I transform plasmid in DH5a E. coli...

17 March 2020 3,375 4 View

Why TOPO-D plasmids with insert are lost in the liquid LB? (being present in the colony)?

I amplified several PCR products and the bands were pretty good in size and unique. The PCR products were inserted into pENTR/D-TOPO, then they were transfromed into E. coli (TOP10 and DH5 alpha)....

09 February 2020 3,411 4 View

Michael J. Benedik

When you do your transformation just include a negative control where you do not add any DNA. If you still see "transformants" after kanamycin treatment without adding DNA, then you know the kanamycin selection is not working well.

Begzodbek Mamajonov

Michael J. Benedik . thanks your help!