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

Hi!

Read Molecular Cloning A Laboratory Manual by Sambrook et.al Volume 2. Chapter DNA Sequencing...., where you get detail information about all size fragments. Now a days NGS has reduced the fragments sizes so no idea whether the new editions contain the information of making total genomic DNA libraries or not

I made total genomic DNA library with bigger inserts up to 23 Kb by using lambda-phage. You have to partially digest DNA so that you get the inserts of bigger sizes when you use total DNA. Try for older version in library read carefully then go as per that.

Good Luck!