Competent Cells Preparation - (Jul/26/2006 )
Why we have to prepare the competent cells at the Optical Density 0.3-0.5?Why can't we use optical density of 0.8?
What is the different between chemically & electroporation transformation?
ok for the first question, it's a problem of growing phases...
When you're at 0,5 cells are growing well relative fast and are very "healthy" (0,3 seems to me little low...)
At 0.8, some part of the culture goes to stationnary phase (not all the culture). These cells are no more suitable for transformation, as they will less srvive the freezing/thawing process (and electroporation is harder than this process...).
So a good OD is a tough point for months of cloning...
For your second question : I would have appreciated that you searched some info yourself...
Anyway : electroporation : better efficiency, you need to desalt your ligation, easier to prepare, not suitable for long vectors and vectors that have an internal possibility of recombination (like repeated sequences)
Chemical transfo : you don't need to desalt your ligation (so no risk of electric shock and loosing some ligation), better for long vectors.
What is the different between chemically & electroporation transformation?
Oh, as mentioned by fred_33, the density at 0.3-0.5 is when the cells are at the log phase of the growth curve for the cells. I suppose u remember that the bacteria growth curve are made of lag phase,log phase, stationary and decline phase. At log phase, " The bacterial population doubles at a regular rate; this is also known as the generation time. The growth rate is rapid, and the rate of cell division exceeds the rate of cell death. Cells produced during this time are very uniform and active metabolically, exhibiting the typical characteristics for the species. "
On the other hand, at "Stationary phase- nutrients are depleted and there is an accumulation of waste byproducts in the medium. Bacteria are less active metabolically. The growth rate slows and starts to equal cell death."
Hence, we use calls at log phase
Those are quoted from here and u can visit for more info.
As for electroporation, I was told it kills more cells but the cells tat are left are mostly positive clones. But for chemical transformation it kills less cells but u have more cells it can hav more background. but it works well. I alwasys use tat cos I don have an electroporation machine, haha
Hope it helps!
If u got time, make competent cells at different optical densities and select the one with good efficiency.
We used to make our cells at 0.8 OD but even if we increased to 0.9, its still good. But this was only for a certain cell type. Some cells r not good at 0.8OD, and u have to grow them at lower OD.
Can you guys share some of your unique protocols for making competent cells?
Check the following articles for chemical competent cells preparation:
Chung, C.T. et al. (1989). One-step preparation of competent E. coli. PNAS USA 86:2172
Inoue et al. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28
Preparing Electrocompetent Cells
1. Inoculate 1L of LB with 10ml of an overnight culture.
Grow the cells with shaking at 37°C until the A600 reaches 0.5-0.7.
2. Chill the culture on ice for 15-30 minutes. Perform all subsequent steps at
4°C.
3. Harvest the cells by centrifugation at 4,000 × g for 15 minutes.
4. Aspirate the supernatant to remove as much of the medium as possible.
Resuspend the cells in an equal volume of ice-cold 10% glycerol. Centrifuge
as in Step 3.
5. Remove the supernatant and resuspend the cells in 0.02X the volume of icecold
10% glycerol. Centrifuge as in Step 3.
6. Resuspend the cells in 0.002-0.003X the final volume (2-3ml) of ice-cold 10%
glycerol. Freeze the cells in 100µl aliquots in a dry ice/ethanol bath and
store at -70°C.
chemical comp cell prep.
1. grow bacteria ON
2. inoculate 1:100 into LB
3. grow until OD600 reaches 0.4-0.6
4. centrifuge at 5000rpm for 5 min at 4C
5. resuspend the pellet in 1/4 original volume ice cold CaCl2 (25ml for a 100ml starting culture)
6. chill on ice for 15 min
7. centrifuge at 5000rpm for 5 min at 4C
8. resuspend the pellet in 1/25 colume ice cold CaCl2 + 15% glycerol
9. freeze the cells in 200 ul aliquots
10. keep at -70C
resuspending pellets are not as easy as it sounds. u need to be careful and gentle with these pellets. no pipetting or harsh shaking.
This one has the advantage of working reliably, at least for us:
http://openwetware.org/wiki/TOP10_chemically_competent_cells
electrocompetent cells :
We used to make our cells at 0.8 OD but even if we increased to 0.9, its still good. But this was only for a certain cell type. Some cells r not good at 0.8OD, and u have to grow them at lower OD.
Hi Malik
I think I agree with you that OD depends on the isolate that you are working with. For eg., when I did growth curve for my Pseudomonas isolate I found that the OD was 0.8 when the total plate count increased. SO i have taken the cells when OD was 0.8. Hope it works.
Has anybody tried making competent cells of Pseudomonas using sucrose medium?
rajitha