Sam:LabNotes/Microbiome-new/2010-1-28

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16S LD-PCR 40cycle amplification for bacteria genotyping[edit]

Backgorund[edit]

  • 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[edit]

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[edit]

  • 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
  • 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[edit]

  • 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