Many problems in genomic library construction - try to construction genomic library (May/30/2008 )

Hi all

I got many problems about construction of genomic library. I use lambda ZAPII phage which predigested with BamHI and treated with CIAP (from Stratagene) as a vector.

First, I extracted the genomic DNA from Mtb using lysozyme/CTAB method. Using this method, I got a high MW DNA but with some smear band. Does it interfere the next step of library construction?

Then, I digested it with Sau3AI restriction enzyme (partial digestion), but It never work well "

I want DNA ~ 3-10 Kb in size, but when I use 1U/1microgram DNA, just 30 sec at 37C, the DNA is cut in a small pieces (l<500-not more than 2 Kb )

How should I do to get the 3-10 Kb DNA fragment ?

Is this DNA size suitable for ligate with phage vector ?

If I want to screen my library with antibody, the insert DNA should be a full-lenght gene for correct expression of protein antigen or not ?

Please give me any comments or share me your experience. thanks so much ^o^

You can lower the temperature to slow the reaction down, which would make it more controllable. You could also switch to BamHI or BglII for the digestion, both of which work better than Sau3AI and will produce longer fragments (though less random). You could shear with a syringe or sonicator to get the correct size fragments, but would thenhave to end repair and use a linker.

you used too much restriction enzyme for partial digestion. you have to make dilutions of your restriction enzyme and make test experiments (dilutions and incubation time) to get which condition can be for you to obtain the target fragments.

Thanks all guys for your valuable comments

After I read your comments, I cut my genomic DNA by diluting the restriction enzyme from 1U/1ug DNA to 0.01 u/1 ug DNA, for 5-10 min.

The results showed that I got my desired DNA fragments. It's a good news for me. So, thank you very much for helping me.