Please suggest me why I am not able to get transformants and how can I troubleshoot the problem?
Initially when I transformed and selected on Hygromycin and G418 plate, I got 5 to 15 colonies per plate but now not a single colony I observed after 5 rounds of transformation.
I checked the incubation and heat shock temp.
I prepared the transformation reagent freshly, such as, TE-Lithium Acetate buffer and PEG.
I used DMSO to increase the transformation efficiency.
I maintained proper cell density during the log phase culture.
now what I should try????
Hello Partha Dey
How much amount of DNA are you loading into transformation mixture? For integrative plasmid 600-800 ng and for PCR cassette ~1000 ng of DNA is recommended per transformation.
Are you using single stranded DNA in the transformation mixture? If so, do you heat up the ssDNA at 95°C for 2 minutes prior to transformation (allowed to the room temperature before addition)?
Heat shock at 42°C for 20 minutes is recommended.
The antibiotic selection plate should be freshly prepared, ideally on the same day of transformation.
Best regards.
When I select for Hygro or G418 resistance I plate my transformed cells on YPD first and incubate o/n then transfer with velvet on drug-containing plate. It helps to the cassette to be expressed correctly. In my hands it helps a lot.
Best
Thank you very much Satyendra Mondal and Marco Geymonat for your valuable suggestion.
Luckily I got the transformants from my last transformation.
There are few things which I tried check and it worked.....
1. the cells density matter a lot. So, I grew the log phase cells till it reach OD600 of 1.0-1.2.
2. During Heat Shock, the Water bath temperature should maintain constant. My water bath showed temperature fluctuation, 42 ± 4-5 °C. Which impacts highly during yeast transformation.
3. I have maintained the proper transformation reaction mixture volume.
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