I am unable to make BL21 DE3 competent cells. I have tried many protocols including CaCl2, MgCl2, and TSS method but every time I failed. On the other hand, if I prepare competent cells for DH5a or XL-blue; i got hundred of colonies in DH5a and even more in XL-blue cells but in case of BL21 DE3, hardly 4-5 or less.
OD600 used for preparation- 0.3-0.4
Vector used for checking competency- pET28b
A couple things to keep in mind with this strain:
- It's an E. coli B strain derivative and all of those are less competent than the ones derived from K12 (like DH5a)
- BL21(DE3) expresses T7 RNA polymerase from the notoriously leaky LacUV5 promoter and if your gene is even slightly toxic this could result in less transformants. Adding 0.5% glucose to your recovery media could alleviate this somewhat because of catabolite repression of the promoter. You could also try switching to a BL21(DE3) derivative that has tighter control like a pLys variant or one that overexpresses lacI.
As far as competent cell methods:
I've had pretty good luck with the Inoue method for BL21(DE3) derived strains.
https://www.princeton.edu/genomics/mcclean/protocols/Inoue-Method-for-Preparation-of-Competent-E-Coli.pdf
(Though I just prepare them at 37 instead of a lower temperature because I'm impatient.)
Hello Vikash,
I agree with Alexandra.I have used the following protocol(long back) and got positive results on DH5 alpha and BL21(DE3). I am copying the protocol for you, hope it works. You can refer to Molecular Cloning by Shambrook et al for more details.
Be very careful while heat shock step. Some people even suggest use of chilled microtips( however I don't know the underlying mechanism)
1. Inoculate a single bacteria from ~12 hrs plate to 50ml LB broth
2. Take OD after 3-54hrs (OD600 ~ 0.4).
3. Keep the culture on ice for about 45’
4. Centrifuge at 5000 rpm for 10 minutes at 4oC
5. Decant all the media and add 30ml CaCl2 solution and keep on ice for 45 minutes and mix by gentle swirling
6. Centrifuge at 5000 rpm for 10 minutes at 4oC
7. Decant all the media and add 1.5ml CaCl2 solution and keep on ice for 45 minutes
8. Add 2 μl DNA in a fresh microfuge tube and add 200 μl of CaCl2 treated cells and keep on ice for 45minutes
9. Keep on water bath at 42o C for 90 seconds and transfer to ice immediately
10. Add 700 LB broth and incubate at 37o C for 45 minutes
11. Mix by gentle swirling and centrifuge at 10000 rpm for two minutes
12. Decant the contents such as about one third of supernatant is left.
13. Mix the pellet gently and spread 100 μl on the LBA plate
14. Incubate at 37o C for 12-16 hrs
Good luck
-Bhuwan
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