Family Recipe - Homestyle Competent cells

I've been getting a lot of random good feedback about my cells. People I haven't even made them for keep asking for them, and quite a few have said they are better than what they have purchased commercially. I think its because I really do make each aliquot with love. The only thing I can think of that I really don't see other people doing is keeping things cold. I make them in the cold room, and aliquot them into tubes that are already in liquid nitrogen. Also, you can keep them in TFB1 longer than 90 min ( I have certainly left them overnight). Maybe I will post the results from this latest batch of ccdb survival cells vs. Invitrogen's, which sell for $189 per ten transformations. I made enough for 100 transformations, essentialy free (1 box =~ $1890 worth of cells) when you borrow all the reagents (just don't borrow everything from the same lab). If they really are that good, I should sell (barter) them for other things I need around the lab, or for my lab courses taught in the city.

Competent cells

Adapted from QIAexpressionist 07/97

TFB1 (Cold) 500ml

100mM RbCl 6g

50mM MnCl_2• 4H₂O 5g

30mM CaCl_{2 •} 2H₂O 0.7g

30mM KAc 1.5g

15% glycerol 75g

pH5.8 w/ 10%HAc

TFB2 (Cold) 500ml

10mM MOPS 1g

10mM RbCl 0.6g

75mM CaCl_{2 •} 2H₂O 5.5g

15% glycerol 75g

pH 8.0 Autoclaved or pH6.8 Sterile Filtered

5M KOH

1. Streak out cells on plate with appropriate antibiotic, incubate 37^oC overnight.
2. Pick a single colony and inoculate 10ml of LB-Antibiotic overnight at 37^oC.
3. Add 1ml overnight culture per 100mL of pre-warmed LB broth containing antibiotic and shake at 37^oC until an OD₆₀₀ of ~0.5 in reached.
4. Cool culture on ice (at least 5 min depending on size), and transfer to sterile round bottom centrifuge tube.
5. Collect the cells by centrifugation at low speed (5 min, 4000x g, 4 ^oC)
6. From this point keep everything cold and on ice!
7. Discard the supernatant and resuspend the cells gently in ICE COLD TFB1 buffer (30mL TFB1 per 100ml of culture). Incubate 90 min on ice.
8. Collect the cells by centrifugation at low speed (5 min, 4000x g, 4 ^oC)
9. Discard the supernatant and resuspend gently in ICE COLD TFB2 buffer (4ml per 100ml culture).
10. Aliquot in 100-200 μl portions in cold sterile microfuge tubes, and freeze in liquid nitrogen.
11. Store cells at -70 ^oC

Tip: Make them with love, and keep them cold, i.e. I hold them next to my heart.