Hi, maybe this is helpful to read:

http://bitesizebio.com/13500/cloning-tips-vector-prep/

http://www.snapgene.com/resources/cloning_tips/

Cheers, Nadine

Thank you miss

:)

We have to distinguish cloning from genomic DNA and cDNA, whether it is from prokaryotes with small genomes or eukaryotes with large genomes, and whether you use libraries with probes, or PCR on naked DNA. I'm also not sure if you mean subcloning or cloning (see below).

1) cDNA libraries are mostly used for the cloning of eukaryotic genes to avoid introns and to take advantage of conditions under which transcripts corresponding to your gene of interest are high. You can use a heterologous gene as probe if you have reasons to believe that there is a good level of sequence homology. If you have purified a protein and you have protein sequence, you can design degenerate primers and amplify a fragment by PCR, which you can then use as probe. Problems occur when transcript levels are low, and when sequence homology is poor. The use of degenerate primers is also tricky. Much of this is now simplified with the rapidly growing list of sequenced genomes, so rather than going through libraries, you can do the preparatory work on the computer and if you have found what you are looking for, you can design specific primers to amplify exactly what you want. Problems with genome sequences are wrongly predicted intron-exon structures. Typically, introns are found at the processing sites of signal peptides or other protein targeting signals, and the prediction programs often decide to go for a longer reading frame which may be the wrong one.

2) Cloning genomic DNA allows you to clone genes even if they are not expressed in the tissue you are looking for, or if they are very poorly expressed (like certain receptors), and they will give you the promoters and 3' untranslated ends as well, but unfortunately also the introns. Genomic clones are very large and the problem is that you may not have complete clones. Again, genome sequencing helps a lot, and oftewn you don't need a library to clone your gene. But the same problem with wrongly pr4edicted intron-exon structures remain. If genomes are very large and polyploid, working with genomic DNA can be a real difficulty. Genomic clones are often isolated after mapping a mutant, and the purpose is to complement the mutation so you have proof that the right gene is cloned. But this only works if the genomic clone is complete.

3) Cloning from bacteria is much easier, libraries can be made in plasmids, and genome sequencing is fast a furious. However, when you complement mutants with plasmid-borne DNA fragments, you can get multicopy suppressors instead of the desired complementors and that's a problem you need to be aware of.

4) PCR cloning can be fast, but you have to be constantly afraid that you might amplify the wrong fragment which will be a contaminant once you have it in the lab. This problem can be overcome by having several PCR primers and carrying out nested PCR, but this depends on how much information you have about the gene you want to clone.

5) And then there is subcloning, that means you have already clones in plasmids, but you want to build new ones. This is much much easier than cloning new genes, and one should not confuse cloning with subcloning (but a lot of people use the term cloning in a very loose manner). If you are interested in problems associated with subcloning (isloating fragments, ligating, making competent cells, designing PCR primers, plan ahead so you can distinguish recombinant from parent plasmids, and all the tricks like blunting, dephosphorylating etc...), then I can answer that in a separate message.

I am working on cloning of mdar gene: we are facing following problems:

1) firstly in isolation of full length gene (earlier unknown) by conventional PCR methods. like RACE.

2) during the transformation of that gene in Vector. because , after transformation it use to give uncertain results.

we are at this stage only so I can tell you this much only... in future if i face other problem i'll tell you.

1) Amplification of the desired gene is a major problem I faced. Taq polymerase could not amplify the gene, so had to use Phusion polymerase. After few days of use, the genomic DNA would no longer give PCR product and had to isolate fresh DNA.

2) getting the correct clone with the desired gene in the vector is such a big problem.

3) plasmid instability is also a big problem

dear researchers, thanks a lot for your valuable information.

Mr. Denecke, yes I wish to know more on problem associated with subcloning . thank you

OK, subcloning into a plasmid.

1) The easiest of all is where you replace a visible DNA fragment flanked by two different incompatible restriction sites (bigger that 200 bp) by another fragment with a different size and flanked by the same two incompatible restriction sites. Typical examples would be to replace a coding region by another coding region in an expression vector. Problems are minimal, but if anything goes wrong it is probably the preparation of the recipient plasmid ("vector"). You cut the vector with the two enzymes, and dephosphorylate the ends before clean up, but even a minor amount of plasmid cut by just one of the two enzymes will give a high self-ligation background and the dephosphorylation never works 100%, so you need to test 1) vector without ligase (to test for remaining supercoil uncut vector), 2) vector with ligase (to test for self-ligation), and 3) vector + fragment + ligase (this is where you want to find your recombinants). So if you have more colonies on the third plate, you've done it, if second and thir plate have equal colonies, it may work or it may not, and if you have equally many colonies on all three plates, then start preparing a vector again.

2) If you clone into a polylinker, cut with two different enzymes, it is the same as the case above, but it is harder because you don't know if both enzymes have cut until you have ligated, transformed and look at the plates the next day.

3) Sometimes you want to put two fragments into a vector at the same time, this works reasonably well when you have three different sticky ends, but of course the efficiency is a little lower than in cases 1 and 2.

4) Much harder is to clone into a single site, unless the dephosphorylation is really good, there will be a lot of self-ligation, and the fragment can ligate in two orientations whilst you may want only one specific orientation.

5) Blunting ends adds another element of difficulty, because nothing is perfect, but it usually works well when the insert is assymmetrical (for instance one blunted end and one sticky end) in cases 1 and 2, but although I have made recombinants in the past with two simultaneous inserts where one of them was blunted at one end, I would call that the pain-limit and encourage alternative strategies.

6) The hardest is subcloning a blunt ended fragment into a blunted vector, but it can be done if you must.

7) Cloning two blunted fragments into a blunted vector requires a amall miracle.

In general, you should always worry more about the vector than the fragment. Fragments can be gel-purified, vector is usually just cleaned up (hence the need for dephosphorylation). If the fragment is large, you may want to do a 4rth control ligation (fragment with ligase alone, to test if you have separated the fragment from the parent vector on gel). The detection limit on gel is 5-10 nanograms, which means that even if you don';t see the contaminant, it can give colonies after transformation.

Oh, and of course you must have good competent E.coli cells. If you don't have good competent cells, then you have a problem. We make them ourself because they are better than those you can buy. We use the calcium and rubidium chloride method and make 500 aliquots every 3-4 months. One of the most competent strains is MC1061, it also grows much faster than DH5alpha and you have nice colonies after 16 hours.

Enjoy :)

Thank you very much sir :)