Hello everyone, I would like to clone a region of about 40 kb, which contains a copy number variation in question, and put this in a BAC or any other plasmid. Does anyone have an idea of how I could do this? I have read papers on BAC cloning, but they suggest random fragmenting of the genome, which is not what I can do. Any idea is welcome. Thanks in advance.
Hi Nivedita,
I am not sure about the 'organism' you are working one. If it is some standard organism (like Human, Mouse, Arabidopsis, rice, wheat and many others), the ordered contig BAC/YAC libraries may already be available with some centres. Get in touch with them and order the clones which you want. Some centres will also screen your probe in their libraries and will send the positive clones.
For other organisms where such facilities are not available...you may have to go by the recommended protocols
bye
Hello Ajay,
Thanks for your reply. I would like to "cut" a specific 40 kb region from some specific breed(s) of cow, and clone this into suitable vector. Alas, I am unable to find suitable protocols for this and was wondering how people manage to clone such large specific fragments without the conventional cut and paste method. Amplifying such large fragments by PCR is not very feasible, so how does one carry on with this?
Hi Nivedita,
For such an organism you may to to see some old papers particularly follow the protocols that were used in very early genome sequencing projects when YACs, BACs and Cosmid (may also be suitable for your work) were routinely prepared. Careful shearing, very mild DNAse treatment or using very rare cutting restriction enzymes etc, could be used for getting large fragments. Get in touch with some centre near your Institute (or in your country) that might be involved in such work. Some researchers would definitely come forward to help you out.
If you want to go by PCR...some kits go till 10-20 Kb fragments or so...then you can sequence them separately. The consideration here will be to use good quality DNA with high integrity (less shearing during isolation)
The other option is go for region specific/limited NGS (see if feasible) to get the sequence of that region in your samples. It may work out to be faster and cheaper. However, this will be limited by how much sequence information is available in the organism of interest.
See what works best for you...it may not be difficult to do as people have done it in past...just choose the right procedure based on the available information of your organism.
bye
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