Help needed in library construction of metagenome - (May/27/2005 )

Hi guys,


I've been having a trouble to clone a heterogenous DNA genome inside a plasmid vector. The heterogenous DNA genome was digested with several blunt-end restriction enzyme to produce varies size of DNA fragments below than 10 kb. Is there any vector that is suitable to produce this metagenomic library?maybe there are certain techniques and precaution to produce a genome library? please help...thank you


Thanks [COLOR=purple]

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I had constructed a soil metagenomic plasmid library with pSK(+),but I used PstI restriction enzyme.why you digested your genome DNA with blunt-end restriction enzyme?In general,the effiency of blunt-end ligation is lower than stick-end fragments.Maybe you have some special intention.I think you should add some suitble linker.

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Could you tell me how large insert in your pSK(+) library? Recently I also prepare to construct a metagenomic library. But I am not sure plasmid pUC19 whether be suit to the construction? And could you tell me your detail protocol? Thank you!

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The higher the copy number of the plasmid you use the more bias you are going to find in your library (pUC19 is very high copy number). Does it matter if you have an unbiased library? If yes then use a single copy plasmid like the BAC vector, if no then any plasmid will do.

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Thanks for Daniel replying!
I had tried to construct a BAC library but failed. Recombiant BAC plasmids were not cut by re NotI. I donot know the reason. First I thought it was the problem of re NotI. Actually it can cut BAC vector into two correct size bands. So why my recombiant BACs can not be cut? Also i had thougt it may be the reason of the purity of BAC plasmids and then i extract BAC plasmids with phenol/chloroform, but the same result. Could you help me analyze the reason? The problem disturbs me for a long time and i cannot perform my experiments further.
Thank you!

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A couple of questions and suggestions:

1. Not I can be difficult to work with. Can you cut your recombinant BAC clones with other RE enzymes? If you add some of the BAC vector to your BAC clones and digest with Not I do you see the vector cut?

2. Were your inserts prepared by Not I digestion?

3. BAC preps can be a quite prone to inhibitor contamination as you have to use a lot of cells for not much DNA. In particular, you can get polysaccharides that are not removed by phenol. Try growing your cells more slowly at 30C rather than 37C as this reduces polysaccharide accumulation.

Cheers

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Thanks!
My inserst were clonged into BamHI-site pBACe3.6 vector and not other single RE enzymes could be used in MCS. I also change other re enzymes only two cut site in this vector but the same results. You thought that there were some contamination such as polysaccharide inhibitoring the reaction. I will try again as your advice. But the accumulation of polysaccharide is enough to inhibtor the reaction in the E.coli cells?

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It definitely sounds like you have a inhibitor contamination problem. Try doing an experiment adding some of your BAC vector to you BAC clones and digest with RE. If the vector does not digest then you have a inhibitor problem.

Yes polysaccharides can inhibit RE digestions if they are in high concentrations. They can be very hard to get rid of, much better to avoid them if possible.

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A CTAB extraction can remove polysaccharides. Another possible solution is to run your RE digests in a very much more dilute reaction. Aim for <1 ug in a 100ul, for example. Also, you could try calling NEB technical support. They are very knowledgeable.

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Hi Daniel,

I have prepared the recombiant BAC plasmids from the E.coli cells grew at 30C and then digested but still the non-cut results. The attachment is the results of digestion analysis. The most left is the control of BAC plasmid and the most right is the marker of λDNA/HindIII, the rest is the recombiant BAC plasmids. Do you think what happened in my experiments? I'm lookinig forwared to your advice. Thank you!

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