I am screening for correct clones after an In-Fusion cloning and transfection in Stellar competent cells. I usually pick up a colony and add it to the PCR-mix (I use HotStart polymerase with an initial activation of 15 min at 95°C). This method used to work with TOP10 cells, but for Stellar cells no amplicons are generated (even with positive clones as confirmed through plasmid extraction and PCR or digestion). However, I also have a lot of negative clones with no clear blue/white screening, so it would be helpful to be able to screen initially through colony PCR.
Hi Stijn,
I prepare a colony PCR lysis buffer (TE + 0.1% Triton-X100) then boil each colony to be screened for 5 minutes in 15 μL of this lysis buffer, then spin down (~13000 G) for 10 mins. I use 2 μL of the supernatant as template for the colony PCR. Never failed me. With that said, I should also say that I patch the colonies to be screened on a new plate, let them grow, and then lyse them. I do this for the sake of organization and, in my case, it prevents false positives (you should do this if you're trying to identify recombinant plasmids).
Hope this works for you!
Hi Stijn,
I prepare a colony PCR lysis buffer (TE + 0.1% Triton-X100) then boil each colony to be screened for 5 minutes in 15 μL of this lysis buffer, then spin down (~13000 G) for 10 mins. I use 2 μL of the supernatant as template for the colony PCR. Never failed me. With that said, I should also say that I patch the colonies to be screened on a new plate, let them grow, and then lyse them. I do this for the sake of organization and, in my case, it prevents false positives (you should do this if you're trying to identify recombinant plasmids).
Hope this works for you!
Hi Daniel,
thank you for the quick reply!! I'll try this out immediately :)
First method: using a pipett tip or toothpick take a single colony, transfer it on a new agar plate (grow the plate at 37C) and put the rest into 30uL of ddH2O (in epp. probe), boil it (95C) for 10 min, cool on ice and spin at 13000rpm for 5 min. Take 1-2uL of the resulted supernatant to PCR mix. Do the PCR, estimate positive clones and take them for miniprep (from plate grown at 37C).
Second method: take single colony, resuspend it in 30uL of ddH2O and take 1-2uL for 20 uL PCR. Initial denaturation should be conducted for 10min (lysis of cells) followed by PCR. Store the resuspended in water cells (on ice) until the PCR products are separated on agarose gel. Take (from possitive reactions) 10uL of water resuspended cell for miniprep.
Good to know the protocol is robust across different cell lines! Good luck with your project.
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