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='''16S LD-PCR 40cycle amplification for bacteria genotyping'''= ==Backgorund== *I identified two possible positive amplicons by 16S-LD PCR validation. However the PCR products concentration might be too low for sequencing. *By doing Size selection + 2nd PCR amplification, I can get enough specific PCR product for sequencing. ==Procedure== Taq-Gold(ABI) recipe 1rxn 4+1rxn --------------------------------------------- H2O 13.5 67.5 10X buf 2.0 10.0 MgCl2 2.0 10.0 Micro Seq primer(f+r,10uM) 1.0 5.0 - Microseq_16S dNTP(10mM) 0.4 2.0 Taq-Gold Enzyme 0.1 0.5 --------------------------------------------- 19.0 95.0 95/5=19 template-1uL '''Template (1uL): 11-1, 11-2, 12-1, 12-2''' 11-1, 12-1: 1uL eliqote from 20uL (E-gel) size selected 1st PCR amplicons 11-2, 12-2: 1uL eliqote from 1st PCR amplicons Program "16S-amp (40cycle)''' ==Results== *1uL 2nd-PCR amplicons were varified by 2% agarose gel **Sample well: 1uL sample + 5uL 0.5X TBE + 2uL 6X loading dye **Ladder: 5uL pre-diluted 1kb-plus ladder [[File:sam012810-2nd PCR.jpg|350px]] *'''The E-gel size selected templates (11-1 and 12-1) showed higer specificity than direct-eliqote template from 1st PCR amplicons (11-2, 12-2)''' *I only use 2nd PCR amplcons from 11-1 and 12-1 for Sanger sequencing. *I purifified the 2nd PCR product(20uL) with Montage filter and sent to Genewiz for sequecing. ==Sequence results== *I got very high quality scores (49 and 50) for both samples. *'''For both sequence, the blast (NCBI nt database) results hit unculturable bacterium''' Sequence File : 11-1-sam012810-16S.seq >11-1-sam012810-16S_A09.ab1 CNNNNANNNNNGNCGNGGNATGATGCNATTTGAACGGACCTTTTTTGNAATAACCCTTCTGGTGGAAATTAGGAAAGGTT AGTGGCAGACGGGGGAGTAACGCGTAGAAAATCTACCTTAAAGACTGGGACAACAGTTGGAAACGACTGCTAATACCGGA TACGCTGCACATAGGGCATCCTAGGTGCAGGAAAAGAGGCCTCTTAACAATGCTCCTGCTTTTAGATGAGTCTGCGTCTG ATTAGCTAGATGGTGGGGTAACGGCTTACCATGGCGACGATCAGTAGTCGGCCTGAGAGGGTGACCGGCCACATTGGGAC TGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCG CGTGATCGAATGAAGGCCTTCGGGTTGTAAAGATCTGTTGACAGGGACGAATAAGCAATGCGAATAGTTTTGTGTATGAC GGTACCTGTTTAGAAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAA Sequence File : 12-1-sam012810-16S.seq >12-1-sam012810-16S_B09.ab1 CNNNNANNNNGGCGNNGTATGATGCTATTTGAGGGGCATCAACTTATGGTGGCTGGGGCTGGTGACCGGACGACGTTTGC GGAAACCGTACGNACCTTCCTTGGTAAGGGGGATAGCCCATAGAAATGTGGATTAATACCCCGTAAGATAGTGGGATGGC ATCATACTACTATTATAGTTACGACGCTTGAAGATGGGTGTGCGTCTGATTAGGTAGTTGGCGGGGTAAAGGCCCACCAA GCCTTCGATCAGTAGCTGATGTGAGAGCATGATCAGCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAG CAGTAAGGAATATTGGTCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGAAGGATGAAGGTCCTCTGGATTGTAAAC TTCTTTTATAGGGGGCGAAAAAAGGGAAATCTTTCTCACTTGACAGTACCCTATGAATAAGCACCGGCTAACTCCGTGCC AGCAGCCGCGGTAA
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