|Topic:||How to improve plasmid DNA transfection efficiency? .|
|Details:||How to improve plasmid DNA transfection efficiency? I have always used 1 µL plasmid DNA (1 µg/µL) during transfection.|
Posted by: Gupta
2017-10-24 21:09:18 MST
| While reading protocol, It look very simple. But you should be very careful with the following points as much i experienced:
1) Check your Plasmid-DNA preparation method ( if mini-perep so use other methods to exclude endotoxins).
2) Optimize tranfection reagents( e.g Lipofectamine 2000, Transit 2020 etc.) first. with maintaing proper cell confluency
3) Reduced serum( Opti-MEM) should not be too old.
4) Do not put antibiotics during transfection in any manner
5) If you are looking for stable transfections, wait little bit more ( 48-72 hour) for changing to complete medim after transfection
6) then start screening with antibiotics
7) Before screening with antibiotics ( need stable one), optimize defined concentration of antibiotics for cell line that you are using.
8) For optimization your DNA, you should first start with 3:1 ( Reagent :DNA) . Then you change the ration in the same way if that cell lines is not showing good efficiency. But at the end, efficiency does not matter much than the goal for which you are looking for sometimes.
Posted by: Mike J
2017-10-24 21:10:40 MST
| If you transfect with Lipofectamine-type reagent, then use the reverse transfection protocol and optimize both transfection reagent quantity and DNA quantity. Typically, OK results are achieved for 1 ug DNA and 3-5 uL of reagent per 200,000 - 1,000,000 cells. Do not use antibiotics at all, this will increase the efficiency.
If you transfect your DNA by electroporation, then use "carrier" ("stuffer") DNA, e.g., herring sperm DNA. Typically, 50 ug of carrier DNA is used and the plasmid DNA quantity an be varied. In electroporation you should also optimize the pulse conditions. Typically, an exponential wave is used with microfarads set at 975 uF, and voltage 180-270V. To increase cell survival after electric pulse use the "cytomix"-buffer complemented with ATP (you can skip glutathione).
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