chemically competent cells - which method do you use? (Mar/10/2006 )
I have to prepare some chemically competent cells and failed at the first attempt. I was using the RbCl2 method and wasn't vigilant enough at keeping them cold. I was thinking of using the method of Chung et al. since it seems easier and the resulting cells can be transformed without heat shocking
1.Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
2.Grow the diluted culture to an OD600 of 0.2 - 0.5.
3.Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is Xml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at 4C).
4.Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible:
1.Centrifuge for 10 minutes at 3000 rpm and 4oC.
2.Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.
3.Resuspend in chilled TSS buffer using 5ml pipet. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
4.Add 100 μl aliquots to your chilled eppendorfs, flash freeze in ethanol/dry ice or liquid nitrogen and store at − 80oC.
1) 100ul cells are mixed with up to 100ng DNA and held on ice for 10 min.
2) Add 0.9 ml TSS/LB/SOC and incubate 37 C for 30 - 60 min before plating.
Is this a good method?
Erm, maybe a stupid question, but do ou heath shock? (you probably do, but it's just not mentioned).
I haven't used this method yet, but no, heat-shocking is not recommended.
See the following:
Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172-5
"One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution."
Chung CT, Niemela SL, Miller RH.
We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.
i have never done using TSS buffer before
we use CaCl2 method and it works fine for XL1 BLue , DH5alpha etc
the overnight culture ( 10ml ) is used to inoculate 50ml LB and kept for growing at 37 degree till
OD600 is 0.5-0.6. Then the culture is transferred to centrifuge tubes and centrifuged at 8000 rpm for five min at 4 degree Cel . The supernatant is removed completely and pellet is suspended in Ice- cold 0.1M CaCl2 . (10ml) . this is kept in ice for 15 min . then again centrifuged for 8000 rpm for five min at 4 degree Cel. again the supernatant is discarded and pellet resuspended in 2 ml Ice-cold CaCl2.
this gives competent cells... store in ice till using it for transformation
and transformation is done using heat shock method
first mix 100microliter of competent cells with 10microliter of ligation mix
keep in ice for 30 min
heat shock at 42 degree Cel for 1 min ( varies with strain of competent cell)
keep in ice( optional)
add LB or media recommended without antibiotics
grow in 37 degre Cel for 2-3 hours ( again depending on ur stain )
and spread it on LB plus appropriate antibiotics plates
keep it overnight at 37 degree Cel
hope this helps
does the TSS buffer work well?/ i am right now in a rut with transforming BL21 cells and the protocol recommends TSS ,, till now nobody in my lab used TSS for BL21 , always used CaCl2 ,, so i wanted to know if TSS is working well or not
all the best
Well it worked.
I grew up a 5ml overnight culture, diluted 1:100 into 50ml the next morning. Grew for three hours, spun down at 1000g for ten minutes, resuspended in 5ml TSS, and froze down in 100ul aliquots. To one aliquot I added 1ng of a plasmid containing an amp gene and left at 4degrees for 25 minutes, then added SOC and incubated at 37 for 45min, plated, etc.
No heat shock was needed.
thank you very much!!
Just want to know.
Does the DMSO in the TSS buffer affect the transformation?
According to Sambrook and Russel, DMSO present as a transformation inhibitor.
And what are the other components used for TSS buffer? =)
TSS transformation is a simple, one-step, reliable protocol that does not require much labor and produces efficiencies of 10e7 to 10e8 transformants/µg of supercoiled plasmid DNA. Transformation efficiencies of 10e5 transformants/µg DNA are possible when bacteria are harvested in late stationary phase or treated with TSS and stored at 4°C for 4 days. Cells may be prepared in advance and aliquotted for storage at -20°C. It is quite handy for preparing large amounts of competent cells for classroom use.
Chung, et al.. 1989 Proc. Natl. Acad. Sci. USA 86: 2172-2175.
C. T. Chung and R. H. Miller 1993 Preparation and storage of competent Escherichia coli cells, Methods in Enzymology, Vol 218, Ed R. Wu 621-627.
Clontech ClonTechniques January 1993:18.
The pH of TSS buffer should be 6.5-6.8, titrate to 6.5 with HCl. Filter sterilize through a cellulose nitrate 0.45 µm filter. Add DMSO. Alternatively, add the DMSO and filter everything through a nylon filter. TSS should not be autoclaved. Store at 4°C. Some lots of DMSO may be inhibitory. DMSO may make some cell lines a little fragile; pipette gently.