I have got the population of Aphelenchoide nematodes. I am trying to amplify their DNA by using different primers as given below:
1. for ITS - 18S: 59-TTGATTACGTCCCTGCCCTTT-39 (forward) and 28S: 59-TTTCACTCGCCGTTACTAAGG-39 (reverse) Vrain et al 1992
2.For ITS- TW81: (5’–GCGGATCCGTTTCCGTAGGTGAACCTGC–3’) and reverse primer AB28: (5’–GCGGATCCATATGCTTAAGTTCAGCGGGT–3’) (Joyce et al., 1994)
3.For D2D3- D2F: 5 ́–CCTTAGTAACGGCGAGTGAAA–3 ́ (forward) and 536: 5 ́–CAGCTATCCTGAGGAAAC–3 ́ (reverse) (Nadler et al., 2006)
4.For 18S- 18SILVOmid F: (5'-CAAGTCTGGTGCCAGCAG-3') and 18SILVOmid R: (5'-GAGTCTCGCTCGTTATCGG-3')
5.fOR D2D3- D2A-F: (5'-ACAAGTACCGTGAGGGAAAGTTG-3') and D3B-R: (5'-TCGGAAGGAACCAGCTACTA-3')(Nunn 1992)
Unfortunately, all these primers didn't work on the DNA of these nematodes. Suggest me the primers for more precise characterization.
Hi, if these primers are already known and used for this organism and they still don't work, have you taken a look into your PCR settings? (Mastermix composition, PCR programme --> especially annealing temperature, proper extension time depending on the polymerase you use etc.). Have you tried a gradient PCR for these primer pairs in the temperature range which might be fitting for them? Do you have problems with unspecific bands or don't you have any bands at all? A not working PCR can have multiple reasons and if you specify the problem and mention what you already tried so far, it is easier to find a solution ;)
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