How much efficiency will remain in commercially available competent cells after one month?
I think usually they last much longer than a month if you keep the cells in -80'c. Transformation efficiency goes down once you thaw the cells but still a workable efficiency. I usually mark the vials that have gone through free-thawing.
Dear Raksha,
Unless you haven't thaw the kit, some supplier claims to retain transformation efficiency upto six months. How about you check one of your competent cell vial for CFU/ug of control DNA and then use the rest of them in your experiments.
I used cells from Promega provided with the pGEM vector system kit
(TA cloning) stored 2 years:) in -70
Hello Raksha,
I have worked with self-made competent cells i.e BL21 and am presently working with KRX competent cells from Promega. As far as the vitality is concerned I had no issues with either BL-21 or KRX. The advantage of KRX over BL-21 are:
1) Higher CFU/ug
2) Pre-induced expression or in general terms the leaky expression that one could see in BL-21 is overcome in KRX system because the T7 pol is under the control of rhamnose promoter which is much more tightly regulated.
3) X-Gal plates could be used to pick out transformants.
As long as you are unaware of the effect of recombinant protein expression in your host, it is recommendable to use a well controlled expression system such as KRX.
You could also look at competent cells from NEB, Life technologies etc..
Good luck..
Yes, I agree with the responses above. It is best to pre-aliquot the cells and keep in -80 freezer. As long as cells do not got through thaw and unthaw cycles, they should be able to work for 3 years. I have kept cells for longer.
Commercial competent cells are very good for experiments where you need highly efficient transformation and where you would usually get very few or no colonies with self-made competent cells. Properly stored (normally, -80 deg) cells are useful for several months for sure. However, if you get skilled in preparation of competent cells yourself, you can do all your work without commercial cells. I find it very convenient and often use commercial ones.
I've been very pleased with the efficiency of commercially available competent cells, especially electrocompetent cells. They are expensive but reliable. I've stored these cells in the -80 C for longer than a month without issue.
We have used electrocompetent cells from NEB, stored them at -80 and they were perfectly working in cloning. The efficacy of the cells didn't change even after several months of storing them in -80. The important thing is to avoid freezing and thawing of the these competent cells because this will affect their efficiency.
HI Raksha, as long as you dont subject the cells to freeze thaw cycles they are good for several years...we have used cells that were 3 years old and we did get high efficiencies of transformation....
I've used commercial cells stored for years without problems; normal and high-efficiency strains kept at -80. Now I make my own cells following the "CCMB80" method based on info from a few patents; the protocol available on OpenWetware works perfectly. Competency for Top10 cells prepared that way is higher than a fresh commercial aliquot for me (head to head comparison done at the same time with same DNA). Back when I made loads of electrocompetent cells they were also fine for over 1 year at -80.
Unless you have designated glassware for making competent cells and really pure water to make designated, pure solutions – it is very difficult to get good competent E. coli cells. Be very careful if you are trying to use glassware in which Saccharomyces was grown – the E. coli would not like it at all. The way to clean the glass from any leftover yeast proteins is to wash them very well, to fill up them with double distill water and autoclave them for an hour. The same is valid for the glassware in which you want to prepare the solutions. Given all the problems to make them – if you have the possibility just get them from a commercial source. I have kept cells for years; they are fine on -80oC. One more thing – you can defrost one tube with 200 l cells and dispense 20 to 50 l of the cells in pre-chilled sterile Eppendorf tubes and use them for regular transformation. This saves some money and works just fine.
Good luck.
Thanks to all for their valuable suggestions.
One thing i want to know how much efficiency of competent cells will be lost if its kept in -30 instead of -80 for more than month.
Thanks
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