A while back I wrote a post on a T4 DNA polymerase dependent ligation independent cloning method. In the comments, Max asked if anyone had a protocol. Since there does not appear to be a simplified protocol available on the web, I thought I would post mine for reference.
It is adapted from a 2006 Organic & Biomolecular Chemistry article by Bonsor et al and although it works for me, I have to caution that I have not been using it for very long so improvements may be possible. If you have any suggestions on how the protocol can be changed or improved, let me know.
The protocol just deals with the bare bones of the digest, T4 DNA polymerase treatment, annealing and transformation. For information in designing the cloning site, see my original article, or the paper.
Here’s the protocol:
Vector Preparation
� Digest: 10 micrograms vector/10U enzyme, 20 microlitres reaction volume for 2hr.
� Separate on agarose gel and extract band
� T4 polymerase treatment
- 0.4 pmol digested vector
- 4 microlitres of reaction buffer
- 4 microlitres of 25mM dTTP
- 2 microlitres of 100mM DTT
- 20U T4 DNA polymerase
- Water to 40 microlitres
- Incubate 30min at 22°C
- Incubate 20min at 75°C (to stop reaction)
� Clean up reaction with PCR purification kit
Insert preparation
� After the PCR reaction, use a PCR purification kit to clean up.
� T4 polymerase treatment
- 0.2 pmol PCR product
- 2 microlitres T4 pol buffer
- 2 microlitres of 25mM dATP
- 1 microlitres 100mM DTT
- 10U T4 DNA pol
- Water to 20 microlitres
- Incubate at 30min at 22°C
- Incubate at 75°C 20min (to stop reaction)
� Clean up reaction with PCR purification kit
Anneal insert and vector
� Combine 2 microlitres insert + 1 microlitre vector
� Incubate at RT for 10min
� + 2 microlitres 100mM EDTA
� Heat to 75°C and cool slowly to RT
� Transform 2 microlitres as normal
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Nick Oswald
Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial Manager of Neuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick on LinkedIn
I’m not sure this is exactly the same thing but have a look at this protocol from OWW:
http://openwetware.org/wiki/Endy:DNA_ligation_using_T4_DNA_ligase
OpenWetWare is a great spot to look for protocols.
Here is a website to help with designing the primers correctly: http://bioinfo.clontech.com/infusion/convertPcrPrimersInit.do
Here is a cool website to help with the design of the primers
http://bioinfo.clontech.com/infusion/convertPcrPrimersInit.do
I kept confusing reverse and forward strand with these strange primers all the time, you’re well advised to use the tool at least to check your primers.
Thanks Max - I didn’t know about the clontech site. When I first tried this method I got very confused with the primers too, so this will useful.
I was thinking about this method and how useful it could be. One of the few hurdles I can see is having to incorporate the ligation-indepent site into various vectors. I can’t help but wonder if this would be a great use of ADDGENE, which was mentioned here in October. If users of this site were to make modifications of the typical (or not so typical) backbone vectors eg pUC19, pBR322 and then deposit them into ADDGENE, this could be a wonderful resource. Another thought is to incorporate Int sites from the Gateway system, allowing easy transfer from the original plasmid into many downstream expression vectors.
Patrick - Depositing modified plasmids in addgene is a really good idea. However, the Gateway int sites would not be required.
Apart from it’s efficiency, the real beauty of this method for me is that, as long as you have the same ligation independent cloning site in each vector, you can put the same T4 DNA polymerase treated insert into many different vectors without modification.
Agreed, you don’t need the Gateway Int sites. I should have been more precise. I meant that there was a second way to generate a bunch of useable backbones quickly. In lieu of re-building vectors for LIC, it would be possible to build just one vector with a LIC site flanked by the gateway sites and then move the desired fragment about this way. The obvious limitation here being vectors with Gateway Int sites are the only useful ones. So… back to the first option.
So now I should get to work and be the first one to deposit something in Addgene for everyone to use. Any suggestions?
Hi Patrick -
The first decision would be which cloning site design to use for all of the vectors, then we could start a program to construct them. Can you email me (hit the “contact” button at the top of the page), so we can talk about this a bit more?
Thanks,
Nick.
Hi Nick I have one question. In the Insert preparation item, you wrote that after treatment with T4 polymerase, 2 min for enzyme inactivation are required. Is this ok? Shouldn’t there say “20″ minutes? Thanks in advance!
Fer
Hi Fer,
Yes, you are right - I have amended the protocol. Thanks for pointing it out
Hi~
I have a question.
Refer to your protocol, you mean Lic is used ‘0.2 pmol PCR product’. Of course I know PCR products conc. But I can not calculate how to unit conversion to pmol.
Hi,
Thank you for your nice protocol.
I have a similar question as above.
As your protocol mentioned, 2pmol of digested Vector and 4pmol of PCR products, we want to know the concentration of the products.
Refer to other paper of LIC, the concentration of the Vector and PCR products are mentioned as ‘ng’.
We are asking you to kindly provied us the exact ‘ng’ of the Vector and PCR products.
Will be waiting your answer.
for dsDNA: pmol x (660/10^6) x N = µg
Where N= the length of the DNA in bp
and 660= the average molecular weight of a base pair
An alternative ligase and restriction enzyme independent cloning method is the USER method from
NEB. The method NEB post on their homepage is not ideal but a couple of modifications have been made (see Nour-Eldin et al, and Geu Flores et al, NAR 2006 and 2007) which makes this method an outstanding method for cloning and fusing PCR fragments. Hope this is of interest to someone.
Hi,
Our group did LIC and we found out 30% percent of the clones turned out to be without insert. Our PI said: the extracted band from the agarose gel still had some supercoiled undigested DNA. Is there a method to separate the linerized fragment from the supercoiled in the digestion? Thanks.
Hi Tigist
This problem is not unique to LIC - it is just that the vector is not digested fully, as your PI has said. Some tips on vector prep can be found here:
http://bitesizebio.com/2007/08/22/cloning-tips-vector-prep/
Another option is to us a positive selection vector, which selects against vectors that don’t contain an insert… see this article:
http://bitesizebio.com/2007/09/06/gene-cloning-positive-selection/
Hi everyone,
I have currently been trying the (S)LIC recombination approach with the subsequent transformation. However, I have been unsuccessful up to now.
I am using a lambda pir based vector and transforming into E. coli CC118 lambda pir. Has anyone tried something similar, or have most people been using the more common E. coli strains e.g. DH5a and DH10B
What T4 DNA polymerase is the best, good enough (reasonable priced)?
Using 10U of T4 DNA polymerase for the PCR insert gets quite expensive.
Hi Joy
I use NEB’s polymerase. I’ve never tried any others, so I couldn’t vouch for them.
Nick
Careful about heating the T4 in the absence of EDTA. It’s a good idea to add EDTA up to a final of 5-10 mM. This ensures the T4 is inactivated before heating. If you don’t, as you heat, the ends will slightly denature before the enzyme is inactivated, and the monster 3′-5′ exo activity of T4 might chew the DNA.
Also, it’s a good idea to do the T4 reactions at 16-20 C, RT at the highest. Once again, this prevents the “breathing” ends from being a problem.
I’ve not done LIC, but this gives me much much higher efficiency when I’m blunting using T4.
Great tip Jeremy - thanks!