Hi, I've transformed my DH5 alpha E.coli cells with pDrive vector with my GOI. After that I plated them on ampicillin agar plate. I got good number of transformed cells (as I am expecting). There is no question that my ampicillin is not working because I did subculture of those transformed colonies into LB broth with same ampicillin antibiotic and cells are growing well. While I cross checked with non-transformed DH5alpha cells in same antibiotic and I found there was no growth. I did many times plasmid isolation from these transformed cells using conventional protocol and I'm getting my plasmid product stuck into the agarose gel wells. I tried for colony PCR and same I didn't get any result there. Now so ultimately I tried to grow these transformed cells into Kanamycin+Ampicillin LB medium, I'm getting no growth at all. FYI my plasmid have both Ampicillin and kanamycin resistance gene. What could be the reason that cells are growing well in ampicillin but no growth I'm getting when I'm using ampicillin and kanamycin together?
Non-transformed DH5alpha cells wont grow in Amp and its normal nothing wrong as they dont have resistant gene for Amp. Also, any anitbiotic Amp or Kana etc is not added for growth of bacteria, but it is added to avoid growth of unspecific or other untransformed bacteria (to prevent contamination). I think you are getting false positive colonies after tranformation. Whats concentration of Amp are you using? ALso why did you add both Amp+Kana both to culture? Is your tranformant resistant to both of them?
You might be getting self ligation of vector thats why you are getting colonies but no results with colony PCR. Please add +ve and -ve control for your tranformation experiments to confirm what is going wrong.Hope it helps.
check your transformed bacteria with comparison with non-transformed on new solid media contain antibiotic. I think its better to re-transform to new competent cells, because clone PCR its not reliable way for checking recombination and other ways to check transformation waste your time
I thikn it is possible that the competent cells you used haven't been made well. Try using other competent cells. By the way, how long did you get colonies after you plated transformed cells on Amp+ plates? If it took to long (I mean more than 16 hr) before visible colonies can be observed, them can't be positive colonies.
XingSheng, It took around 12 hrs when I got colonies on my plate, well I did again transformation of same plasmid with same insert again I have got good number of colonies on ampicillin agar medium but again When I'm doing subculture of those colonies in ampicillin and kanamycin LB medium they are not growing but I'm getting good growth in only ampicillin medium.
I have a similar issue with S. aureus transformants. The transformants grow well on chloramphenicol containing plates but when I grow them in a liquid media contains the same concentration of chloramphenicol they don't grow.
Hi, can I ask why bacterial can growth on LB agar containing ampicillin which plated with competent cell?
But there is no bacterial growth on LB agar with or without ampicillin plated with transformant
Hi, I had a similar problem. I transformed bacteria after ligation of a digested pLKO.1 and mcherry insert. Bacteria grew on amp LBagar plates and I obtained a normal number of colonies per plate. I did a PCR screening and te majority of the colonies were positive for my insert. Howver, when I grew them in liquid LB medium they didn´t grow, has anyone any idea why this can be happening?
Anaïs Notario I have the same problem where my bacteria grew well in LB agar plates but not in liquid medium. Although I see a relatively small amount bacterial growth in the media. But I would be interested to know the reason too. Did you get any better results or did you find any solution?
Hi
Do not plate the cells immediately after the transformation. Keep it at 37 for 1 hour in antibiotic free media before you put it onto the plate containing antibiotics. Give the time to plasmid to express antibiotic resistant genes.
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