I have to construct a genomic library (bacterial DNA) by using the technique GLPCR (Genomic Library PCR) who can guide me? Shall I digest DNA and vector (plasmid) by various enzymes? I would like some guidance on the steps I have to do.
In my opinion you can construct the cDNA library for your GLPCR which is the master source of your future work.
Gateway TOPO Vector should be preferable.
This can give you a speed for the functional analysis i hope so in your case------
Good luck
Manoj
I would suggest for genomic libraries of unknown origin - to go for what is popularly known as Shot gun cloning. you can sonicate the genomic DNA down to a specific clone'able size, eg. a mean distribution of 1kb or less. Then use universal adaptors (available from commerical sources or part of your GLPCR kit) which allows u to ligate dsDNA to the sonicated fragments followed by PCR. The mixture is then cleaned up and cut with a restriction enzyme that cuts in the adapters to allow for sticky end cloning of the fragments into libraries using an appropriate vector. Usually we use low copy pBR322 origin plasmids for libraries to account of instability of genomic fragments once cloned. The other alternative is to go for TOPO cloning of TA cloning vectors directly. The adapters will help as primer sites or identification markers if you are doing Next Gen sequencing of the libraries.
dependiong on what can of genomic library youwant to build , you fill find the answer in Current Protocols of Molecular Biology. The old Maniatis might still be of some help but the era of Lambda GT 2 , Lambda GT3 is far away now
Restriction Digest the genomic dna with enzyme like ecoR1 and slect the size of 15 kb or above. Gel extract the bands and precipitate with 2 propanol and use for cloning in abcterial plsmid such as Puc18 or pFOS depending on size of the insert or to reduce the number of clones...gud luck
Thank you for your suggestions. I used pbluescript vector, i obtained on agarose gel several DNA fragments after digestion of the DNA of interest with three enzymes (Eco RI, EcoR V and Pst I), knowing that the plasmid vector was digested by the same restriction enzymes. Now the next step is cloning, What should i do? Since i have several DNA of interest, shall i ligate all the Dna with the vector digested by the corresponding enzyme ? What about screening by the GLPCR method?
Invirogen and clonetech have genomic library kits but always it depends on the desired size of the insert.
In my opinion, generation of genomic libraries has become an old fashion in these days.
Just sequence the entire genome and amplify your target gene by PCR. You will save time and money.
Good Luck
I agree with Mahmoud, the library prep for Next Generation Sequencing is very easy (Nextera XT kit) but I guess it depends on what you want to do with your library ! Let me know if you want more info on NGS. Good luck!
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