6 replies to this topic

[Clare]

Hello

In my old lab, when transfecting 293Ts we used the following method...

In brief:

- Culture cells in chloroquine for 30 mins
- prepare transfection cocktail (structural vector, sample vector, envelope vector, Calcium chloride)
- Add 2X HBS to the cocktail (containing 50mM HEPES, 10mM KCl, 12mM Dextrose, 280nM NaCl, 1.5mM Na2HPO4)
- add to cells and viola! Beautiful GFP expression the following day.

Now that I am in a new lab (on the other side of the world) I am trying to set up this method again. BUT I have had no luck so far

Has anyone used this method that can offer advice on making up the solutions? I have successfully used this method zillions of times, however, in my old lab someone else had made the solutions (and I can't get in contact with them!!!)

I know the pH of the HBS is critical so the other day I did a series of 8 tranfsections with different pHs, but did not see the GFP that I used to

The structural/env vectors I actually got from my old lab. Everything else is from the new lab.

Hope someone can help

Clare

[miBunny]

Fresh calcium chloride can help.  Make sure that it is not too hydrated (it picks up water over time so that you end up adding less calcium and more water).

The pH of the 2X HBS is the biggest issue and the problem could be the pH meter.  Is the probe in good shape (not too old and relatively well maintained)?

Check pHs over a range (every 0.05 units).  

Getting a fine precipitate matters.  Are you bubbling the mixture for 15 seconds?  a small 1 mL pipet really helps for getting the precipitate fine enough.  Are you seeing the small little black spots evenly spread on the bottom of the plate.

I personally never used chloroquine.  We didn't seem to need it to get 100% of the 293Ts to turn bright green.

[Clare]

Hello

The calcium chloride I used was fresh as I opened it for the first time.
The pH meter is new (well, about 1 year old) and when I did my test transfections I tested a range of different pH of the HBS (from 6.80 to 7.30, every 0.05). I saw a little bit of GFP at pH 7.1, 7.15 and 7.2.
Yes, I bubbled the mixture and I saw the little black dots. In fact, the cells looked exactly as they should (from when I did the experiments during my PhD) except no bright GFP

Thanks for the reply I'll attempt it again with some fresh 293Ts and see how they go. I seed 4 million cells in a 10cm plate prior to transfection (ie: about 5-6pm the previous day). How many do you seed?

Clare

miBunny, on Aug 19 2009, 01:21 AM, said:

Fresh calcium chloride can help.  Make sure that it is not too hydrated (it picks up water over time so that you end up adding less calcium and more water).

The pH of the 2X HBS is the biggest issue and the problem could be the pH meter.  Is the probe in good shape (not too old and relatively well maintained)?

Check pHs over a range (every 0.05 units).  

Getting a fine precipitate matters.  Are you bubbling the mixture for 15 seconds?  a small 1 mL pipet really helps for getting the precipitate fine enough.  Are you seeing the small little black spots evenly spread on the bottom of the plate.

I personally never used chloroquine.  We didn't seem to need it to get 100% of the 293Ts to turn bright green.

[Clare]

PS:
What's the final conc. of CaCl2 that you use? I was going to order some solution just in case mine is dodgy. My protocol says use 144ul of 2M CaCl2 in my transfection cocktail and I can't seem to find one that concentrated!
Clare

[miBunny]

I use the same amount of calcium chloride.  I always made mine from powder.

I had another thought....  Are your cells free from mycoplasm contamination?  That can really ruin your transfections!

[Clare]

I am sure they are ok - I got them from a colleague in the same building who showed me lovely GFP expression in her cells.

Hmm...shall try again next week and see if things improve!
Clare

miBunny, on Aug 20 2009, 01:59 AM, said:

I use the same amount of calcium chloride.  I always made mine from powder.

I had another thought....  Are your cells free from mycoplasm contamination?  That can really ruin your transfections!

[miBunny]

Try her plasmids. If she's doing Calcium phosphate transfections, see if you can borrow her reagents and then do a substitution of each of her reagents with one of yours (using the best behaving HBS solutions).  This may let you figure out which of your reagents is the problem.

Good luck!!!!