We are trying to generate an infectious clone of a plant virus called cassava brown streak virus in 3 fragments of 3-4 Kb in size (virus is ~10Kb) but have been failing to get more than one clone into the TOPO-XL vector. Cloning individual fragments is not a problem but trying to add 5' fragment to the middle fragment or vice versa has been very difficult to say the least. Any tips and sharing successful protocols will be much appreciated.
You could try Gibson cloning, you will have information on NEB website, or see this
https://www.neb.com/~/media/Catalog/All-Products/0AA961B294E444AFBEDD5C4A904C76E6/Datacards%20or%20Manuals/manualE2611.pdf
This methods allows you cloning large fragments.
Hope this helps
You could try Gibson cloning, you will have information on NEB website, or see this
https://www.neb.com/~/media/Catalog/All-Products/0AA961B294E444AFBEDD5C4A904C76E6/Datacards%20or%20Manuals/manualE2611.pdf
This methods allows you cloning large fragments.
Hope this helps
I'll second Raditijo's suggestion above to use a fosmid. I was lucky with KOD extreme from Novagen, my fragment was over 15kb.
You could try restriction free cloning. The In-fusion kit from Clontech is really good: http://www.clontech.com/GB/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid
http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web&cd=2&sqi=2&ved=0CDcQFjAB&url=http%3A%2F%2Fwww.clontech.com%2Fxxclt_ibcGetAttachment.jsp%3FcItemId%3D17497&ei=GvFXUsCKOeew7AbBwoGACQ&usg=AFQjCNGe_-AepCmzhyeXCW5GPhd5TDa3og&sig2=9SeHchaIuPUCLRJdVmKuOQ
As Raditijo suggested, fosmid could be an idea vector for the purpose!
I guess one can use a 2-step cloning strategy - to make two frgaments with known restriction sites and then ligate them together!
I recommend using: pTZ57R/T Cloning vector (Thermo Scientific, USA)
This size is similar as HIV. What we did before for HIV is as following:
1. Get the overlapped fragments (each one for 3-to-4 kb, about 1kb overlapped, so we started from four but not three fragments)
2. Using Taqase to make the fusion one by one (I used fusion, actually, the overlapped size is larger than regular fusion)
3. Check the sequence.
Recently, I used the Gibson Assemble from NEB as Anne Coléno-Costes listed above.
But I used it for only two fragments once(actually I used it for insertion of the fragment without the ER). I prefer to this if I have to make the clone about 10k again.
I have not tried the TOPO-XL.
I think the easier is to amplify the entire 10kb fragment using a log expand polimerase. Several comercial (such Roche or Fermentas) sells this kind of enzime that amplifiy without problems 5-25 kb targets. Then clone it in a vector that supports large fragments. For me is easier to clone PCR fragments adding a restirction enzime target in the 5' of the primer plus 4 or five nucleotides to allow cutting near the end. After obtain the PCR product you must cut with this enzime and clone at the same site in the vector choosed
Dear all, many thanks for all the suggestions, some we have tried already such as amplifying virus as a single fragment without luck but some methods are new to me so we will give it a go. Best wishes - Maruthi
Try Lambda phage you can happily clone fragments up to 23 Kb or slightly more.
I worked and made Genomic Library. It was fine.
Hi Marhuthi
We also make infectious clones (geminiviruses) and it is a real pain in the backside. We are currently also trying the Gibson kit, cloning two fragments into the vector that has also been created by PCR, but no success thus far. What vector are you cloning the virus into? Will it be a dimer or partial repeat?
Sending you this link by Life Technology, I have not used might be helpful for your question http://www.lifetechnologies.com/us/en/home/life-science/cloning/topo/long-fragment-cloning.html
Marhuthi,
In my experience fragment assembly can vary tremendously from one sequence to another. I have used both the Gibson kit and Infusion assembly approaches, and their efficiency is very much dependent on sequence complexity. Did you perform any sequence optimizations on your CBSV sequence prior to synthesis? When you say your assembly is not working does that mean you are unable to get correct clones or that you are not getting any clones at all? If you are not getting correct clones then perhaps there is selection pressure due to toxicity of the final product on your TOP10 cells (I am assuming you are using the TOP10 competent cells that come with the kit). Are you using electroporation or chemical transformation? My assumption is that you were probably using a Golden Gate Cloning approach initially to assemble your fragments, since you stated individual fragment cloning was not an issue. If you have already attempted to directly amplify the full fragment with no success then its most likely your sequence has several pockets of secondary regions that may be interfering with the polymerase, which could be alleviated via sequence optimization if that is an option. Alternatively, you can use a sequence analysis software to determine where those complex regions are on your sequence and partition your fragments so as to avoid designing overlaps or junctions in those regions. I hope this helps.
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