If yes how would you test the transfection if you haven't got a fluorescent microscope .I have used AMAXA kit for doing this, so please do advice if there are some points to keep in mind.
Thanks
To test the transfection:
1) if you transformed with a fluorescent reporter you can use FACS or microscope
2) If there is no fluorescent reporter you can do a Western Blott with an antibody of the protein or the tag (V5 or His Ab)
3) if no Ab available, you can do a PCR on cDNA with T7 and BGH primer to amplify specifically your mRNA of the vector to check the expression
4) If really you can not do that (but consider to change lab!) PCDNA3.1 is usually a G418 resistance, then by using 1mg/mL of G418 on HEK-293, only those with the vectors will survive.
PS: despite your experiment never forget: EMPTY vector, Control vector (tagged to check the efficiency), no vector and your vector of interest
Attention: H-293T (antigen T) are resistant up to 6mg/mL of G418, it is not possible to obtain stable with this antibiotic....
Rq: Using AMAXA on HEK is like use a Hammer on an egg. Lipofectamine LTX provide 100% of transfection on this kind of cells
To test the transfection:
1) if you transformed with a fluorescent reporter you can use FACS or microscope
2) If there is no fluorescent reporter you can do a Western Blott with an antibody of the protein or the tag (V5 or His Ab)
3) if no Ab available, you can do a PCR on cDNA with T7 and BGH primer to amplify specifically your mRNA of the vector to check the expression
4) If really you can not do that (but consider to change lab!) PCDNA3.1 is usually a G418 resistance, then by using 1mg/mL of G418 on HEK-293, only those with the vectors will survive.
PS: despite your experiment never forget: EMPTY vector, Control vector (tagged to check the efficiency), no vector and your vector of interest
Attention: H-293T (antigen T) are resistant up to 6mg/mL of G418, it is not possible to obtain stable with this antibiotic....
Rq: Using AMAXA on HEK is like use a Hammer on an egg. Lipofectamine LTX provide 100% of transfection on this kind of cells
I am new to this subject. I will do the stable transfection to overexpress my target gene in pcDNA3.1 (G418 selection) to HEK293. Could you please send me your usual protocol. Do I need to do the killing curve to check the effective dose of G418 and linearize my plasmid before tranfection?
Thank you so much for your help!
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