Hello everyone,
I have a recombinant clone (My gene fragment, 2 restriction enzyme sites and pet 28a vector) that gets synthesized by a company. Then I added 20ul DEPC water to that 5ug clone and stored it in a -20 deep fridge. After that, I ordered a Thermo Scientific Competent BL21 cells kit. Afterwards, I added 3ul of my clone into 50ul of competent cells, incubated it in ice for 30 mins, heat shock it for 30 sec, then again incubated it for 2 mins, and put the cells tube in a shaking incubator for 1 hour. After 1 hour spread the cells (containing clone) on LB+agar plate containing ampicillin. The next day, a few cells grew in that plate and for the confirmation of the transformant I performed Colony PCR, but the colony PCR results were not good and bands didn't appear.
Kindly let me know what to do next or how to confirm the transformant.
3 uL plasmid reconstituted in 20 uL water, added to 50 uL cells sounds like a lot of DNA (not sure of your exact condition). Usually adding a few nanograms of DNA is enough and too much DNA would only reduce transformation efficiency.
Have you verified your electrophoresis condition, PCR condition and confirmed no secondary structures in your primers for colony PCR? Usually I directly proceed with Sanger sequencing, as colony PCR cannot detect possible mutations.
Changing the cell line might also help, but might be a later step in the troubleshooting process. Good luck!
Did you add any nutrient broth (LB/SOC without antibiotics) to the vial before the 1 hour shaking? The cells are frozen in a salt solution without any nutrients so you need to add those for the cells to grow.
several reasion.i,e poor DNA quality, incorrect vector design, improper transformation conditions.
Quick question, did you check to make sure the cells your ordered and received were chemically competent and not electro competent? My lab received electro competent cells by mistake and those really don't work well for chemical transformation.
Also, you could try using a control vector, something that you already have on hand of a similar size to your new vector and with amp resistance. Transform them side by side and see what you get for each.
Amy Klocko Yes ma'am, the kit I ordered gave the cells the SOC medium. I've added 250ul of SOC medium to the cells before keeping the cells in shaking incubator.
Amy Klocko Yes maam I've ordered BL21 competent cells with catalogue number EC0114. They also gave the standard DNA so that we could check whether the cells were getting transformed or not.
Yesterday we also performed IPTG induction but we only got blue colonies no white colonies.
Kindly help
Leran Mao Yes Sir, the PCR conditions are already standardised. Please suggest how much amount of DNA could be added for transformation.
Sandeep Kaur
Nano gram range should generally be alright.
I frequently reconstitute 5 ug DNA in 200 uL nuclease free water, then add only 0.5 uL or less to 50-100 uL cells. Also watch out for your media and amp stock - they should be relatively fresh and unexpired.
Do you not get white colonies in your DNA or the control DNA? As recommended by other researchers, I also recommend you try the control plasmid to avoid cell-related issues.
Leran Mao Thank you for your valuable suggestions.
I'll check the stock of media and antibiotic.
I am not getting white colonies in my clone DNA (my gene of interest).