Protocol Online logo
Top : New Forum Archives (2009-): : Cell Biology

transfection is not working - (Sep/04/2010 )

Hello everyone,

I had a problem with transfection. i am trying to transfect VSV-g plasmid with Gp293 Luc cells (retrovirus VLP producers) using lipofectamineTM2000. I tried this on 10 cm dish.

I ran agarose gel with my DNA. its very good.

But my transfection is not working, i donno why??? i tried several times. i synthesized DNA two times. None of them were working.

i used below Protocol for transfection:

cells were seeded on the day before transfection in the ratio of 6*10^6 cells/ dish.

On the day of transfection the cells were 90% confluent.

24 micrograms of DNA was added to 1.5 ml Of DMEM(with out serum)and gently mixed it with pipette.

60 microliters of Lipofectamine was added to another 1.5 ml of DMEM (with out serum) and gently mixed it with pipette and incubated it for 5 min.

after 5 min DNA with 1.5 ml of DMEM was added to lipofectamine in 1.5 ml of DMEM and gently mixed it with pipette and incubated it for 20 min.

in the meantime media in the 10 cm dish was replaced with 9ml of fresh DMEM with out serum.(here in this step i tried with DMEM with 10% FBS also)

After 20 min 3ml of the lipofectamine-DNA complex was added to 10 cm dish and incubated at 37degree centigrade , 5%co2 incubator for 2 days.

On the 2nd day HEK 293 cells were seeded on 6 well plates in the ratio of 1*10^6.

After two days virus supernatant was collected using 0.45 micrometer filter and 100 microliters of the virus supernatant was infected on HEK 293 cells( before infection media was changed with serum free media), after 4 hours of infection media was changed back to serum media.

Luciferase assay was performed after 48 hours.


Thank You
Sravi.

-Sravi-

- 24ug is a lot for a 10cm dish
- 60ul is also a lot, it is not recommended for Lipo 2000
- for Lipo 2000 better change media after 12 hours
- I culture only 2x10^5 cells

have you checked presence of virus after just 24 hours? We check transfection efficiency just after 24h max. we never go for 48h, but we do not produce virus, we use pFLAG plasmids.

do not change media after infection, let it stay.
do not filter your virus supernantant

-Curtis-

THANK YOU SO MUCH FOR UR REPLY

Really?? am i adding DNA and lipofectamine in high quantities???? But, i am following the protocol given by invitrogen(http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf).

How to check if there is virus or not after 1 day????

This time i am planning to do transfection on 6 well plate. can u please tell me how much amount of DNA and lipofectamine i need to add?????
and which ratio i need to seed my cells.


Thank You
Sravi

-Sravi-

Sravi on Sun Sep 5 18:31:25 2010 said:


THANK YOU SO MUCH FOR UR REPLY

Really?? am i adding DNA and lipofectamine in high quantities???? But, i am following the protocol given by invitrogen(http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf).

How to check if there is virus or not after 1 day????

This time i am planning to do transfection on 6 well plate. can u please tell me how much amount of DNA and lipofectamine i need to add?????
and which ratio i need to seed my cells.

Any other suggestions???? plzz post


Thank You
Sravi

-Sravi-

Is this the first time you perform transfection? If yes, I would suggest that you start with smaller plate, like 24-well plate.

The transfection efficiency of every cell line is different. For difficult to transfect cell line, include PLUS reagent in your transfection with Lipofectamine will sometimes improve the transfection.

Also, I found that if you use OptiMEM, the transfection efficiency does different from the transfection performed with serum free RPMI or any other medium.

For transfection with Lipofectamine, I always change my medium after 5 - 6 hours incubation with the DNA-lipid complexes.

You may also want to try other transfection reagent. In my hand, jetPRIME from Polyplus Transfection does transfect better than Lipofectamine LTX with PLUS reagent.

Good luck.

-virusfan-