Hi All,

I am interested to hear everyone's view on the various cloning techniques, such as In-Fusion, LIC, SLIC, Cold Fusion, CPEC and in vivo cloning. Could you share your experiences with these techniques and why you would prefer one over another?

Best regards,

Linda

Hi Linda,

I personally still prefer In-Fusion because there is no need to 'process' the insert except for a simple purification step  (we use AMpure). The cons- vs.LIC and SLIC is obviously the price (although ClonEZ is also the same enzyme but much cheaper) and vs. Gateway is the fact that re-cloning is not conservative (applies to LIC and SLIC also) due to re-amplification of the inserts.

For all of these I would recommend inserting a (negative) selection marker-ccdB, sacB, lacz etc. , if you really want  to make your own vector suite, so that non-recombinants are easy to spot...

An important point that seems to get lost in the In-Fusion vs. LIC vs.SLIC  argument is that a vector designed for one system can be used for any other as they all rely on the production of 5' overhangs (by whatever means) so the different vector suites that have been developed should not be seen as mutually exclusive with regards to 'system' -only exclusive by primer extension required.

Nick

Hi Linda,

I agree with Nick regarding the compatibility of (S)LIC vectors. There are no major differences between the requirements of those and it's a matter of costs. All of the steps can be automated if necessary. We use the SLIC vector set that were constructed by Sabine's and our group using the ccdB selection cassette which is a BIG advantage since you don't get any background colonies (similar to Gateway). Ocassionally you don't get ANY colonies but at least there are no false positive ones. :-) Seriously once established the system is very convenient compared to restriction based cloning..

Here's how we do it : http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/lic/index.html

I would also suggest to have a look at the RF cloning publication published by Tamar Unger's group which also can be used with any of the vectors designed for Infusion or (SLIC) or in principal ANY vector (PubMed ID: 20600952). 

I hope that helps.

Best regards

Hüseyin

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Dear Linda,

as Hüseyin mentioned, we have developed a strategy and also generated a list of vectors that we are willing to share (named pCoofy for CoreFacility) I am just preparing the manuscript and hopefully we can publish them soon. If you are interested I can send you the list of vectors and also the MTA. We have integrated ccdB with a strong promotor, there is zero background. And you don't need any reagent (only recA or T4 Pol, costs very little)

As we based the E.coli vectors on EMBL pETM vector series and as Hüseyin contributed the pFastBac versions and the Sumo backbones it is a common Max-Planck - EMBL MTA.

Many departments in-house and also outside institutions (EMBL etc) use our vectors and protocols. It works in their hands as well, we have a lot of positive feedback.

A question back concerning your insect pipeline:

we also wanted to use flashbac due to deletions, time saving and now the UNIC enhanced version. However, we still don't have a permission from OET that we can propagate the virus, so we would have to buy there virus constantly. How are you using this ?

Hi Sabine,
Hi Hüseyin,

I am still using restriction enzymes for my cloning (shame on me?) and I am really interested in changing to SLIC. I was wondering if it would be possible for me to get some plasmids from you. Do you mind sending me the list of your pCoofy vectors and the MTA? Is it also possible to get the plasmid for expressing His-3C protease?

Hüseyin , you mentionned the E. coli BW23474 strain on your website, could you tell me where I can order it from?

Many thanks,

Patrick

Dear Sabine,

Thanks for sharing. I will get in touch via email on those vectors.

Answer to your question regarding OET flashBac:

I doubt OET will allow others to propagate their virus based on discussions I had with them. However, we made a deal on bulk buy. Also, we have developed the transfection protocol for 24 well plate. This means for each transfection/recombination reaction, we use only 1ul (20ng) flashBac DNA. It's not expensive considering the amount of time saved. Using list price for calculation, it's less than AUD $20 per reaction. Negotiate with OET and each reaction can be less than AUD $10.

Hope this helps.

Best regards,

Linda

Hi Nick, Hüseyin and Sabine,

Thanks for sharing. Any comments on cloning efficiency with your method of choice? 100% positives? What about effect of insert size?

We used various techniques including RE for our construct generation. It's not an ideal operation and I am hoping that we can decide on one. We have tested all the techniques that I have listed earlier but the result is not conlusive when I start to consider insert size, % positive and cost. We haven't tried putting in a selection marker...

Thanks,

Linda

Dear Linda,

thanks for info on FlashBac

With our pCoofy vectors we obtain between 1 to 200 clones per trafo. In 263 cloning reactions, 95 % were succesful. Over the whole size range of inserts 150 bp - 3900 bp we have on average 20 clones / trafo.

Best wishes

Sabine

Hi Linda,

In-Fusion with the pOPIN vectors was giving OPPF 94% (of PCR products) success rate for cloning  when I left and I've never done the maths here but use it for just about everything (new vector construction etc...). Size does not seem to be an issue-in Oxford and here in Barcelona we have cloned everything from 100b.p. to >5k.b. With the largest sizes we see a drop in efficiency but that is really because we do not standardise the moles/copies of insert going into the reactions so we if we use 100ng of 100b.p. we should use 5ug of the 5k.b. insert but of course no-one ever does!

With regards to FlashBac compatible Bacmids-we use the academic version from Ian Jones in Reading-not Cathepsin/Chitinase deleted but very efficient for recombination and simple to grow and linearise.

Also the pOPIN and pPEU vectors are pTriEx- based so you can do the 3-host screening (coli, mammalian and baculo/insect) with the same miniprep and still have the choice of In-Fusion, LIC or SLIC!

Good Luck

Nick