transfecting HeLa cells for the first time... - help needed....please (Nov/09/2005 )

I have ordered Lipofectamine from invitrogen and purified my plasmid DNA's , CsCl equivalent.

Have never done any tissue culture before, please help me as i have limited time..

We will be getting the HeLa cells stock, stored in Liq. nitrogen,

1. can any body let me know the medium to start with thawing, and later on after transfection (with or without fcs) ?

2. which one is prefferable.. 6-well plate or 24 well plate?

3..concentrations of DNA for transfection?

4..can any body expalin me what is the use of trypsin-edta whileculturing the cells and whats the protocol for it?

I am sure i will have more basic questions, once i hear from any one of u ...

thanks..jhil

Hi jhil,

Don't worry, HeLa cells are very easy to grow. I have grown HeLa in both RPMI-1640 medium supplemented with fetal bovine serum (FBS) (10%), L-glutamine (2 mM) and MEM (minimum essential medium) (Eagle) with 2 mM L-glutamine, Earle's BSS and 10% FBS. During transfection, no antibiotics should be used.

24- or 6-well plate? depending on your purpose of transfection. If I am going to analyze protein, I use 6-well plate, although people have used 24-well plate for western.

3..concentrations of DNA for transfection?
Follow the instruction coming with Lipofectamine

4..can any body expalin me what is the use of trypsin-edta while culturing the cells and whats the protocol for it?
It is for lifting and subculturing cell. For protocol, look here
http://www.protocol-online.org/prot/Cell_B...ture/index.html

1. can any body let me know the medium to start with thawing, and later on after transfection (with or without fcs) ?

DMEM middle or RPMI middle ih 10% serum and peni strepto are ok. For transfection i use same medium but with NO serum and NO antibiotics. But recently, vairus did a post (see here) saying that results for him goes very good with a complete medium during transfection


2. which one is prefferable.. 6-well plate or 24 well plate?
i thnk 6 well plates are better because for subsequent experiences you'll have more cells.

3..concentrations of DNA for transfection?
concentration of DNA is not the major point. It's the mass of DNA to transfect which holds the flame. In general, 1µg for a well from 6-well plate is ok. And ratio DNA to lipofectamie is also case sensitive. I've done experiments and found that with my preparations, a 1:3 ratio DNA:lipo is better.

4..can any body expalin me what is the use of trypsin-edta while culturing the cells and whats the protocol for it?
you gonna find this protocol on the protoocol online cell biology field. But in general it's at 37° with gentle agitation before and after. you have also a dicsussion on Wash cells or not after trypsinization

Next week, I'm going to transfect Hela cells again with and without serum during transfection to see which one is more efficient. For expression plasmids with a fluorescent reporter I got good results with serum during transfection (low toxicity and high efficiency) with Lipofectamine2000 (and also with Fugene6, which was just a little less efficien). For my purposes, I need maximum efficiency, so I'm going to compare with and without. I'll keep u posted...

thank you all !!

Varius, please keep me posted about ur results

today i did the ligation, hopefully i 'll get some positive clones and then i will start preparing for transfection.

wish me good luck...

let me read some more and i am sure i 'll have more questions,

jhil