transfection : lipofectamine2000+BHK21 monolayer +pcDNA4A -help....

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raboo
raboo's picture
transfection : lipofectamine2000+BHK21 monolayer +pcDNA4A -help....

hi,
can anyone has related  transfection experience - any tips, suggestion are welcome..i will be doing the exp. in coming time...
1. lipofectamine2000- amount needed .....
2.BHK21 monolayer - any special treatment  etc. to improve uptake....
3.pcDNA4A  T/A  LIC vector (+1.4 KB insert)- amount needed per well, time-before rt pcr screening etc....
(primers were supposed to be dephosphorylated - what is that... I just ordered regular primers from IDT... does that  mean i will have to CIAP treatment to vector or primers?....)
4. does anyone have any idea that pcDNA  is efficient as dna vaccine- for mice i mean...
thanx

Jason King
Jason King's picture
Primers do not usually have 5

Primers do not usually have 5' phosphate groups. If you are just doing a PCR detection with these primers then I don't think it would matter whether they were phosphorylated or not. If you want to clone the PCR product into a T/A vector then you wouldn't be needing to dephosphorylate the vector so also wouldn't need to have 5' phosphates on the PCR product.

Regarding Lipofectamine, I would start off using the manufacturer's suggestions. This will mean doing about 3 different ratios of DNA to Lipofectamine - but don't re-invent the wheel unless you need to.

Good Luck.