Not sure I understand your question. BL21 is used for expression of recombinant protein. However for preparing recombinant plasmid that carries a lentivirus component, you can use DH5a. You may want to check with customer service at GeneCopoeia. They use DH5a for recombinant plasmid prep. pdias

I used DH5aplha for lentiviral plasmids transformation. I would recommend it.

Which plasmids you are planning to clone? I did Rev, VSV, Tat and Gag-pol. I got good quality and quantity on Maxi.

Hi, Peter. yesterday I wrote to GeneCopoeia, let's see which response I will get.

Hi, Ranjan, we've just got the pLKO.1 from Addgene (http://www.addgene.org/10878/). They suggest us to use the Stbl3, but I've seen that they also suggest the DH5alpha for other plasmids with the same backbone.

My experience is that DH5a is not an optimal host for plasmid with highly repetitive sequences (e.g. LTR of the lentiviral vector). I'd suggest XL1Blue or Stbl3.

I agree with Kin Lam Fok. We have had problems in the past with DH5alpha and lentiviral vector production as well as HIV-1 molecular clone production. My suggestion is Stbl2/Stbl3 as they keep the integrity of the plasmid over a longer period of time.

I would advise the same.....use Stbl3 instead of DH5alpha. Our maxipreps of lentiviral constructs with DH5alpha were so poor in yield, less than 100ng/ul!!!

It is worth the extra money you pay for the Stl3 cells.....

Good luck!

V.-

Alexis

I agree with Kin Lam, Michel and Veronica above in that the yields of recombinant plasmid are somewhat low with DH5a. However I did not find this to be a problem for tranfecting into the packaging cell line. For the various genes I tried, the packaging line produced high lentiviral titers in the supernatant. I had to dilute the virus containing supernatant for transformation/infection of the the target cells. Besides, one typically needs to transfect the packaging line only once and then collect the the 'assembled' lentivirus containing sup for transformation/infection of the numerous target cell lines. The kit that I used for purifying the plasmid after DH5a transformation was the QIAGEN EndoFree Plasmid Maxi kit (#12362). They do larger kits. Just my thoughts, although I do not claim to be an expert on LV transformation/transfections/infections. I do not work for any of the companies I quoted.

Peter

The Maxi we get from DH5alpha cultures (500 ml) is in the range of 4000-6000 ng/ul. I use the same kit #12362 from Qiagen. Most of my plasmids are same backbone as you mentioned (pLKO.1). The packaging cells we use is 293T. Our lentiviral supernatant work great and we even do not concentrate it. for GFP, mCherry, and serveral other genes of interest we cloned everything in DH5alpha only. The DH5alpha clone #18263-012 we buy from invitrogen.

If you need to make any changes in your protocols or get further advice you can always email me.

I am very grateful for all your advices! That is the kind of clues that I (a "lentibeginner") was looking for. These advices will help me to not get in much trouble before getting the good results. I did not think twice.I just ordered the Stbl3 cells.

Despite that you guys think that the MAIN trouble when using the DH5alpha with this kind of vector is the plasmid concentration, the cloning inefficiency or some nucleotide change/mismatch?

Ranjan, just for curiosity: you think the DH5alpha is the better choice when using a "non-LTR" backbone or you could also use the Stbl3 with this kind of plasmid (as you mentioned for GFP and mCherry)?

You can use DH5a to amplify the lentiviral plasmid DNA but generally the DNA yield is lower (or sometimes much lower e.g.5-10 folds) than using Stbl3. However, Stbl3 is endA+ so a lot of time you get endonuclease co-purified with your miniprep DNA and you see smear on the agarose gel after restriction digestion but good DNA yield (no more than 2-3 hr digestion). We usually get 30-60 ug lentiviral plasmid DNA from Stbl3 in 6-7ml LB per miniprep, The yield will be around 5-20 ug if using DH5a. We rarely use maxiprep DNA to package the lentiviruses, 30-60 ug DNA from Stbl3/miniprep is good enough.

These informations about packing plasmids are new for me. Thanks Pooja.

Hi everyone, I have recently used Stbl3 cells, I ahve seen a significant improvement using lentiviral plasmids. i was able to recover positive plasmids inside some of my colonies culturing them at 30C. My LB medium is (10gL of Tryptone, 10g/L of NaCl, and 5g/L of Yeast ext). I am a bit worried about 2 things at the moment, What salt NaCl concentration for LB medium is better for Stbl3 cells 10g/L (high) or 5g/L(low)? Or does it work well with either one? Is there another way to remove endonuclease after DNA purification with mini/maxi preps besides phenol/chloroform? THanks

HI @Pooja Panwalkar! What final concentration (NaCl g/L) of salt is consider low in LB?

In our lab we frequently prepare retrovirus and lentivirus plasmids in DH5alpha. We have not identified any problems with recombination  (verified by sequencing, viruses infect cells and produce gene products), though we have not compared side-by-side with a Stbl3 strain. So certainly DH5alpha can be used productively. Curious what parameters actually contribute to any differences in yield in DH5alpha vs. Stbl3??

I have spent at least 6 months to get a good pLenti6.3 plasmid by using Stbl3. Here I wanna share some of my experiences about pLenti and Stbl3. As above mentioned we have to use low salt con. 5g/L, culture should be grown just 8-10 hr only otherwise plasmid yield/quality will be poor. 

I followed above things so finally I got good plasmid(+gene), but the transfection efficiency/virus quality is poor when compared to vector, still I have no idea why the efficiency is varying, though I could see equal quality of plasmids on agarose gel. After selection with selection marker I could achieve stable cells. 

Thanks everyone for your  more valuable information about Stbl3 and pLentivirus.

Thanks,

-Uchur

Hello everyone! I found your answers helpful, although I would like to make some inquires myself for the some affair. I'm currently doing my MSc Thesis and I have spend  4 months to find out what to do with the lenties!!! 

The project is to insert a big fragment (6kb) into a pWPI with blunt ends (owning to dam methylations to the insert from XL1blue bacteria). I didn't succeed it and now I'm trying to insert a small fragment of 500bp but with no results. I use DH5α* bacteria for transformation. After the inoculation the incubation temperature of colonies is 37C as for the plates.

*Unfortunately there is no one who has Stbl3 in the other labs and we I don't have the time frame to order

Could anyone please provide me with, or suggest me any protocol for lentivirus cloning in DH5α bacteria? 

We switched from Stbl3 to NEB Stable and got better results regarding the recombination problems mentioned above (You can easily make competent cell stocks. No need to buy every time). These cells seem to be useful for cloning of repeat rich sequences.

https://www.neb.com/products/c3040-neb-stable-competent-e-coli-high-efficiency#tabselect0

Hi all. I feel like the general opinion of this discussion is that STBL3 should be used for lentiviral constructs, because of higher yield and less recombination between LTRs. Does anyone have a genetic explanation for that? As far as I know DH5a and STBL3 are both recA1 (or recA13, which is a less stable variant).

Hi,

Please dont over grow the any culture for pLenti viral vector amplification.

Catch the culture with in 8-10hrs, you will get good yield and quality as well.

Finally, Stbl3 is the ideal strain for lenti vectors.

Thanks.

@ Johann, I also have been confused by the genetic explanation for Dh5 vs stbls.  I know from functional experience with my own hands that stbl3 is better for unstable constructs.  This link below has some info but I am sure it is not required to always use stbl3 for lentis unless they are bigger or the gene is instable. Genes over 10kb in general seem to fall apart more easily, we grow at 32 and ease up on the shaker speed.  Multiple hairpins and repeats are always a problem.  I still have no clue on the bacterial genetics tho.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563744/

I'm surprised by the near-uniform consensus on the advantage of using Stbl3. Does anyone have DATA (please attach and show) to show for Stbl3 vs. DH5alpha for growing plasmids containing LTR sequences?

Our lab has used DH5alpha routinely for retroviruses/lentiviruses containing LTRs successfully over and over, never once observing problems with recombination as far as I can tell by sequencing or on a gel.

Dear all, I have lenti viral shRNA plasmid , shall i use DH5 alpha for multiplication purpose...can any one share your experience.... waiting for your precious reply....

Dear all, thank you for all the questions. I have the same question than Alexis because I have a new lentiviral vector, pCW-Cas9, and Addgene recommends to transform the plasmid into Stbl3. Does anyone transformed this plasmid into DH5alpha bacteria?

I have worked with a few different lentiviral vectors. In my experience, some of them can be propagated in DH5a without problem, but not the others. That's why some people above were puzzled by why special strains are needed while they never had a problem in DH5a. Even with the same vector, the orientation or context of inserts between the repeats can make a big difference in the difficulties handling them. A rule of thumb is to always grow cells at 30 deg to avoid troubles, though you will likely get lower yields compared to 37 deg C growth (about 50%). Stbl3 and NEB Stable are both great and can be home-made competent at high efficiency with general competent cell-making protocols (I use the low temp long time growth protocol), so you don't need to BUY them again if you don't want to.

Irfan. Where do you have this information from? I absolutely believe that you have tested it and that you got the results that you describe but according to databases and the manufacturer description DH5a is RecA1, meaning Rec negative.

http://ecoliwiki.net/colipedia/index.php/DH5_alpha

https://tools.thermofisher.com/content/sfs/brochures/710-021761-B%20Comp%20Cell%20Fin.pdf

Dear Alexis,

Please find your answer below: 

FAQ: Is NEB Stable a substitute for Stbl2 or Stbl3?

Yes, NEB Stable is recommended in all difficult cloning experiments.

The genotype of NEB Stable is not related to the Stbl2 or Stbl3 cell line but the performance of NEB Stable is superior.

The performance of all 3 strains was tested by cloning of a DNA fragment with 5 x 32bp repeats. The test construct is named pUC-5xREP and it was created using the Gibson Assembly method of cloning. Following transformation of the Gibson Assembly reaction into the respective strain, 5xREP insert stability was analyzed by colony PCR:

Stbl2 colonies achieved 1-mm diameter in approximately 24 hrs at 30°C. This population of colonies all carried plasmid clones with deletions (33 of 33 colonies analyzed).

Stbl3 colonies achieved 1-mm diameter in 18-20 hrs at 30°C. Of 33 colonies analyzed, 22 contained full-length 5xREP insert (67% correct).

NEB Stable cells grow well at 30°C and 1-mm diameter colonies are obtained after 18-20 hrs at 30°C. 5xREP insert stability was best when using NEB Stable as the cloning strain (97% correct, 32 of 33 colonies analyzed).

Importantly, NEB Stable carries the endA mutation (in contrast to Stbl3) so that isolated plasmids are free of Endonuclease I.

Ref: https://www.neb.com/faqs/2014/02/27/is-neb-stable-a-suitable-substitute-for-chemical-competent-stbl2-or-stbl3

https://www.neb.com/faqs/2014/02/27/is-neb-stable-a-suitable-substitute-for-chemical-competent-stbl2-or-stbl3

Thanks to every one..

Don't learn the hard way. Lentiviral plasmids can get very screwed up when propagated improperly (like, in DH5a instead of NEB Stable or Stbl3, or if you overgrow your bacteria). Recombination can be an extreme pain to figure out, so just buy the right bacteria, and culture at the right temperature, gang!

There have been a lot of answers here focused on yield, but I want to reiterate some of the other answers that mention recombination. The Long Terminal Repeats (LTRs) have a tendency to recombine, so you can easily lose your insert or get something funky. Stbl3, XL-10 Gold, and similar cells are highly recommended for lentiviral plasmids because they greatly reduce the frequency of this recombination. (In an effort to speed things along, I recently transformed an LV plasmid with DH5a while waiting to receive Stbl3 cells. The transformation was great, but I had lost the insert. Stbl3 is the way to go.)

Brian A Pryce , thank you so much for bringing this up. I used the original bacterial stock from Sigma and extracted the lentishRNA plasmids. In fact, the knockdown efficiency was very nice. However, when I was trying to amplify a little bit more plasmids using DH5a, I've done 3 times of infection and I didn't see any knockdown. What you said might be what I've encountered here.

Meanwhile, I've tried the PvuII enzyme digestion as Sigma suggested and found no issues. That's why I guess, either the recombination or the modification by DH5a abolished the expression of my target shRNA.

Thus, I believe we should try to avoid any unexpected issues and stick to Stbl3.

Not a good idea. Not only plasmid yield from DH5a is low, you have high risk of lose the plasmid or have truncated plasmid probably because of recombination. Strongly suggest Stbl3.

Not a good idea. Yield is low and construct gets messed up. Coming from personal experience.

Lets say we don't have Stbl3 and can't acquire it too in the near future as well, and we are stuck with DHFa, what conditions are recommended to amplify a plasmid like pCW57.1 (LOWER TEMPERATURE? LONGER INCUBATION?). Kindly assist. Thanks.

Yes, But...

When using lentiviral vectors, Stbl3 has advantages. If your lentiviral vector is big, then Stbl3 for sure will be better to use. Stbl3 have higher transformation efficiency with larger plasmids, and also have less mutation and recombination rates.

But, I want to remind you that DH5 alpha was the standard for transformation for many many years, and it used to work. Using Stbl3 will just make it easier for you to pick up a fewer number of correctly recombined clones.

If you only have DH5a then try it. Most probably it will work, but if your plasmid starts behaving weird, then Stbl3 may save your day.

Not a good idea, Stbl3 are excellent for growing viral vectors with LTRs because they are RecA deficient.

We recently generated plasmids in DHFa and also produced viruses from pCW57.1 backbone and the system is working. Be sure to remove the ccDB (toxic gene) from the backbone.

Good luck.

This is very confusing. All of the materials related to amplification of AAV/Lentivirus plasmids contaning LTR clearly say that strain with RecA- should be used and DH5a shouldn't be used because it is RecA+, but from genome description it is actually RecA- (RecA1 mutation). Instead Stb3 or TOP10 strain should be used, but both DH5a and TOP10 has RecA1 mutation. So they are the same in this respect.

"RecA1 is an inactivated form of RecA. RecA1 is deficient in all known function of the RecA gene specifically in ATPase activity, binding with DNA in the presence of ATP, and changing conformation in the presence of ATP and repressor cleavage. Cells of this genotype are UV sensitive due deficiencies in DNA repair mechanisms.

Why select this mutation in E. coli lab strains? For reduced occurrence of unwanted recombination in cloned DNA."

https://2014.igem.org/wiki/images/5/58/Genetic_backgrounds_of_each_Escherichia_coli_strain_used_in_The_ST2OOL_Project.pdf

DH5alpha - F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–

Stbl3 - F- glnV44 recA13 mcrB mrr hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1

TOP10 - F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-

Could anyone clarify this?

Stbl3 has RecA13 which I only found as "RecA13 differs from RecA1 in the lower stability of inserts."

https://2014.igem.org/wiki/images/5/58/Genetic_backgrounds_of_each_Escherichia_coli_strain_used_in_The_ST2OOL_Project.pdf

Addgene recommends Stbl3 as RecA-

We stick with Stbl3 at 30-28C culture temp, it keeps our plasmid features (repeats loxp , HS4 Ins, >18bp Restriction enzymes repeats ) intact compared to other cells. For minipreps we use a culture vol. of 5-10ml. this yields about 25-50µg of DNA, the wash step is repeated x2 always. Maxiprep derived stbl3 DNA has been used for in vivo transgenesis without sequence errors or repeat region recombination. I do have to say, that following transformation you should pick up single colonies and PCR check repeats sites, later screen good candidates clones and sequence. Those that pass the screening criteria work well. Best.

Many lentivirus plasmids deposited at addgene were using NEB stable cells. The advantage of NEB stable vs stbl3 is that NEB stable is endA-. It is not worth using DH5alpha as it may increase the risk of recombination.

NEB stable Genotype

F' proA+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR)/ ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14-  Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS-mcrBC)

I have used NEB Stable and I recommend using it instead of Stbl3. In my experience the plasmid yield is higher and purity is more. As mentioned earlier that there might be a bit of smear in Stbl3 purified plasmids. I do not recommend using DH5a for any repeat containing plasmid i.e. lentivirus or AAV plasmids. There is high chance of repeats being affected as well as recombination occurring.

For pcW57.1, I have used DB3.1 strain which is ccdb resistant for amplifying the plasmid. For cloning you need the ccdb gene and hence, there is no point in amplifying the plasmid without it.

I have used DH5a for generation of a construct with pLVX series backbone using gibson assembly, and never got a right colony following transformation of the reaction into the respective DH5a. Sequencing showed recombination occuring in every colony between 5' LTR and 3' LTR. However, it is confusing that the amplification of pLVX series backbone goes very well (validated by sequencing).