Any tips for critical parameters in the Gateway LR reaction?

06 December 2016 5 2K Report

I'm struggling with the LR reaction for a long time.

1. I'm using the pENTR4 entry plasmid with a human MKK5 insert - it is sequenced and OK.

2. I'm using pLIX_403 destination vector (Addgene #41395). I have checked it 4 different ways: (1) Restriction mapping - was OK; (2) Ampicillin and Chloramphenicol resistance - was OK; (3) ccdB functionality - no colonies grew out from TOP10 or from Stbl3 cells when transfected, and (4) functionality of the Tet repressor-P2A-PURO cassette - transfected the plasmid into HEK and cells became PURO resistant.

3. After the LR reaction a lot of colonies grew out, and no colonies grew from the pLIX403 destination vector only. However, when I check the plasmids on a gel, the recombined plasmids (lanes 4-8 on the attached picture) are much smaller than the expected size (one light band at ~4kb and one at ~2.5 - 2kb instead of around 9.1kb) compared to the empty vectors (lanes 2-3, size around 9.3kb)

4. I know that lentivirus plasmids are prone to recombination. The same thing happens when I transfect the cells into Stbl3 cells instead of TOP10 and/or culture them @30oC instead of 37oC.

5. I'm wondering that anyone else has met with this problem? I tried another lentiviral vector (pINDUCER21) with the same result. Can anyone help me with some suggestions? Are there any critical parameters which I'm missing either during the LR reaction or during transfection or plasmid isolation?

Thanks for the answers in advance

Monika Gostic

I have no experience with Lenitiviral vectors, but I have performed LR recombination on many occasions and it was always successful. I'd recommend the further:

- Make sure your att recombination sites are actually capable of recombining! ( sometimes the att site is named in a certain way but the sequence is different - I have used the original sequence of the vector and checked in ApE plasmid editor for the sequence)

- Sequence not only your insert in your entry vector but also a bit of entry vector with it

- Make sure of the correct plasmids and the orientation of inserts with restriction reaction after isolating them

- Use the high quality LR enzyme and follow the protocol (incubation over night etc)

- The ratio between entry and donor vector should be 3:1

- Add fresh TE buffer to reaction

Hope that helps! Good luck

Alex Agrotis

Hi Domokos

I have also been using gateway cloning to make lentiviral expression vectors. I have found that they are unfortunately extremely prone to recombination, in my mind more so than other LV vectors, most likely due to the LTR sequences in addition to the attB sites which are also repetitive. The destination vector alone is not prone to recombination because you select against this with chloramphenicol, and the resistance gene is present in between the likely sites of recombination.

Nonetheless, it should be possible to clone your desired construct. You just have to be quite stringent with screening clones by restriction digest and doing everything on mini-prep scale. Luckily for me, I usually only need around 1-5 µg of plasmid to make enough LV for my experiments, so if you need to scale up then you may run into difficulties.

Here is what I do:

LR reaction for 4 hours (generally according to manufacturers protocol), then transform into NEB stable competent cells (New England Biolabs). Go for the most efficient transformation protocol.

Grow plate overnight at 37°C or longer at 30°C.

Pick a large number of colonies (at least 6 per construct) and grow 3ml liquid culture overnight (or longer at 30°C). Note that on your plate there may be a mixture of large, fast-growing colonies and smaller colonies that take longer to appear. Pick a mixture of both, but in my experience the slower-growing colonies contain the correct construct.

Use entire 3ml culture to do plasmid extraction. Some cultures may show disappointing levels of growth compared to others, but still go ahead with your mini-prep protocol and you should get a bacterial pellet. You may need to vortex the culture tube thoroughly as I find the bacteria are often quite sticky.

Elute plasmid in ~30 µl of water or elution buffer. You might find that some plasmids give a very good yield and others less so, however the low yield plasmids are often correct.

Screen plasmids by restriction digest to confirm correct size and lack of additional unexpected bands, then sequence positive clones if necessary. Pool the positive mini-preps if you need a larger amount of plasmid for an experiment.

If you need to re-propagate a plasmid that you have already made by this method, then don't rely on glycerol stocks or re-innoculation. Instead do re-transformation and screen colonies again to avoid recombination. Let me know if you want any details on specific steps. Good luck!

Domokos Bartis

Thanks for the answers, both are very useful.

Monica, what is the composition of the TE buffer you use? I used invitrogen's standard TE buffer, 10mmol Tris, 1 mmol EDTA, pH 8.5

Liu Linli

Hi, I also meet the same problem, after LR reaction, the plasmid size is smaller than except, I use the pLgw EcoDam-V5-RFC1 as the destination vector(10.2k) do the experiment as protocol and my fusion plasmid except size is around 10k ,but after I check the size is around 6k, I don't know how to solve this problem, could you give me some advice? thanks a lot! @Domokos Bartis

Molishree Umesh Joshi

Hi. I have been facing a similar problem. I used Mach1 cells for transformation. Usually this cell line grows very rapidly and we dont see as much recombination. However, with the LR reaction all the colonies were recombined. Growing at 30C did not help. Thanks Alex for sharing ideas to troubleshoot this.

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