can you clone the pcr product and sequence with vector primers or am I not understanding the question?

I didn't clone the sequence. After Gel Extraction PCR product was being sequenced and only primer portions were found matched. The probability of non-specific binding may be diminished by reproducible Sharp band after gel electrophoresis. 

I am now planning to clone the sequence. I have an inquiry that will i do cloning just to get the better sequence result or what else? Thank You @Paul Rutland

Are you saying that you got a much bigger band than primer-dimer and that it sequenced ok but that at each end the primers were correct but that in between there was a good sequence but the sequence did not match your target DNA Avizit?. If this is the case then you have the primer sequence that amplified the target in your sequencing  so you can blast these exact primers ( not now degenerate in the amplimer )  and also blast the whole sequence between the primers. It may be that you have a contaminating DNA which amplifies better than your study DNA ( for instance the plant DNA that the fungus was growing on perhaps ). If you do have a good sequence then cloning is not useful as you probably have enough sequence to identify where the problem is and design a way round the problem. Possibly nested pcr where you enrich the target DNA with quite good primers and amplification then have internal nested primers to re-amplify a smaller part of your target after the first round pcr has given you a few different amplimers but that would really be a very small genome for the nested primers to anneal to and amplify and 30 cycles of the first pcr would have enriched the target area by about 1000 million times. If you do try this route remember that the second round pcr needs very little target (1/10th ul of product) and only 10-20 cycles of pcr as each 10 cycles makes 1000 times more product and if there is too much product it self-anneals and the amplification is very messy

paul

Absolutely @Paul. For Genomic DNA i have done PCR with ITS1 and ITS4 primers. That time DNA seemed ok. Then PCR reaction was done with my gene specific primers where i have found a sharp band of size 378bp. and surprisingly  only primer sequences were found matched from both end that was confirmed after BLAST (Where Query Coverage was only 12%). I did BLAST with the amid sequences between primers which showed no similarity. Even primer length was large (24bp).

I did PCR (re-PCR) with the yielded PCR product but that time i use same set of primers to check whether that sequence was really amplified by the primer or not. Nested PCR will be cool i guess too. But if i get nested pcr result positive what would be the interpretation. 

One thing to mention that, My target gene sequences for fungi are not very much reported. (only 2 or 3 sequences are present in Data Base).

Thanks Paul.

I am slightly surprised that the match is only 12% as 2 x24mer primes is 48bases out of 378 and randomly 1 in 4 bases will match of the rest but did the primers blast to lots of places one of which amplifies much better than ITS perhaps. The value of nested pcr is that assuming that your sequence amplified quite a lot but not as well as the 378 baseproduct then reamplification with nested primers which anneal to ITS but not to the 378 product may still amplify ITS as it may have been enriched in the first round but not quite visible yet. It is hard to know what is happening when genome sequence is not available ( pseudogenes gene families, copy number problems are possible) but I suspect that eventually you may have to use a different first primer set possibly 5 or more bases into the its sequence which should not amplify your 378 sequence as only the primers have homology to 378.

If you have some sequence of ITS in your fungus you could theoretically try inverse pcr where you restriction cut the dna,ligate the ends to form circles and pcr out in both directions from the known sequence to generate more sequence into the introns but that may be difficult. hopefully more genome sequence will become availablesoon good luck Paul

 Paul i am afraid that i am not sure u r getting me right. As i am working with Fungi DNA i did check the DNA with ITS sequence and found a good quality of DNA i am working with. Then i did PCR with my gene specific primer (Not ITS) using that DNA to check the presence of that gene in that fungi species. BUT i surprisingly found only primer sequence matched. I will try nested PCR. Inverse PCR will also be cool.

Thank you so much Paul for all of your plausible and solid suggestions. I hope i will get you in my side in this very beginning of my research life and so forth.

yes you are right Avazit I did misunderstand what you are amplifying but I hope that the advice still holds for your GOI. Gene specific primers are a problem as gene sequences are highly conserved whereas intra genic and intronic sequences are less important so not conserved so are more useful in designing primers as they are less likely to work between species which is why I thought inverse pcr ( if no intronic sequence is available for primer design) might be an option although there are technical difficulties with this. Perhaps a primer set to a different part of your GOI woulhappy to help if you think I cand confirm its existance ...might be worth a try

good luck

paul

Thank you sir...

I will work in that way..