Sam:LabNotes/Microbiome-new/2010-1-28: Difference between revisions

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>Sam Chiang
>Sam Chiang
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  11-1, 12-1: 1uL eliqote from 20uL size selected 1st PCR amplicons
  11-1, 12-1: 1uL eliqote from 20uL size selected 1st PCR amplicons
  11-2, 12-2: 1uL eliqote from 1st PCR amplicons
  11-2, 12-2: 1uL eliqote from 1st PCR amplicons


==Results==
==Results==
Line 32: Line 31:
*'''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)'''
*'''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 only use 2nd PCR amplcons from 11-1 and 12-1 for Sanger sequencing.


==PCR product purification with Montage PCR filter==
==PCR product purification with Montage PCR filter==
Line 42: Line 40:
*Centrifuge the filter at 1000xg. for 2 min to retrieve the purified PCR products.
*Centrifuge the filter at 1000xg. for 2 min to retrieve the purified PCR products.
**'''The concentration purified PCR products were about ~40ng/uL in 20uL (by Nanodrop, blanked with 1X TE)'''
**'''The concentration purified PCR products were about ~40ng/uL in 20uL (by Nanodrop, blanked with 1X TE)'''


==Sample dilution for Sanger sequencing at GeneWiz==
==Sample dilution for Sanger sequencing at GeneWiz==
Line 54: Line 51:
**Step2: Mix 1uL original 16S-microseq primer-f (100uM) with 19uL H2O -> 5uM, get 5uL of diluted primer (-> 25pmole)
**Step2: Mix 1uL original 16S-microseq primer-f (100uM) with 19uL H2O -> 5uM, get 5uL of diluted primer (-> 25pmole)
**Mix the solution from Step1 and step2 -> 15uL, transfer into a 8-well stripe tube. Label the sampe on the tubes.
**Mix the solution from Step1 and step2 -> 15uL, transfer into a 8-well stripe tube. Label the sampe on the tubes.


==Sequence results==
==Sequence results==
*I got very high quality score (49 and 50)for both samples
*I got very high quality score (49 and 50) for both samples.
*'''For both sequence, the blast (NCBI nt database) results hit uncultureable bacterium'''   
*'''For both sequence, the blast (NCBI nt database) results hit uncultureable bacterium'''   


Sequence File : 11-1-sam012810-16S.seq
Sequence File : 11-1-sam012810-16S.seq
 
>11-1-sam012810-16S_A09.ab1
>11-1-sam012810-16S_A09.ab1
CNNNNANNNNNGNCGNGGNATGATGCNATTTGAACGGACCTTTTTTGNAATAACCCTTCTGGTGGAAATTAGGAAAGGTT
CNNNNANNNNNGNCGNGGNATGATGCNATTTGAACGGACCTTTTTTGNAATAACCCTTCTGGTGGAAATTAGGAAAGGTT
AGTGGCAGACGGGGGAGTAACGCGTAGAAAATCTACCTTAAAGACTGGGACAACAGTTGGAAACGACTGCTAATACCGGA
AGTGGCAGACGGGGGAGTAACGCGTAGAAAATCTACCTTAAAGACTGGGACAACAGTTGGAAACGACTGCTAATACCGGA
TACGCTGCACATAGGGCATCCTAGGTGCAGGAAAAGAGGCCTCTTAACAATGCTCCTGCTTTTAGATGAGTCTGCGTCTG
TACGCTGCACATAGGGCATCCTAGGTGCAGGAAAAGAGGCCTCTTAACAATGCTCCTGCTTTTAGATGAGTCTGCGTCTG
ATTAGCTAGATGGTGGGGTAACGGCTTACCATGGCGACGATCAGTAGTCGGCCTGAGAGGGTGACCGGCCACATTGGGAC
ATTAGCTAGATGGTGGGGTAACGGCTTACCATGGCGACGATCAGTAGTCGGCCTGAGAGGGTGACCGGCCACATTGGGAC
TGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCG
TGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCG
CGTGATCGAATGAAGGCCTTCGGGTTGTAAAGATCTGTTGACAGGGACGAATAAGCAATGCGAATAGTTTTGTGTATGAC
CGTGATCGAATGAAGGCCTTCGGGTTGTAAAGATCTGTTGACAGGGACGAATAAGCAATGCGAATAGTTTTGTGTATGAC
GGTACCTGTTTAGAAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAA
GGTACCTGTTTAGAAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAA
 
 
Sequence File : 12-1-sam012810-16S.seq
Sequence File : 12-1-sam012810-16S.seq
 
>12-1-sam012810-16S_B09.ab1
>12-1-sam012810-16S_B09.ab1
CNNNNANNNNGGCGNNGTATGATGCTATTTGAGGGGCATCAACTTATGGTGGCTGGGGCTGGTGACCGGACGACGTTTGC
CNNNNANNNNGGCGNNGTATGATGCTATTTGAGGGGCATCAACTTATGGTGGCTGGGGCTGGTGACCGGACGACGTTTGC
GGAAACCGTACGNACCTTCCTTGGTAAGGGGGATAGCCCATAGAAATGTGGATTAATACCCCGTAAGATAGTGGGATGGC
GGAAACCGTACGNACCTTCCTTGGTAAGGGGGATAGCCCATAGAAATGTGGATTAATACCCCGTAAGATAGTGGGATGGC
ATCATACTACTATTATAGTTACGACGCTTGAAGATGGGTGTGCGTCTGATTAGGTAGTTGGCGGGGTAAAGGCCCACCAA
ATCATACTACTATTATAGTTACGACGCTTGAAGATGGGTGTGCGTCTGATTAGGTAGTTGGCGGGGTAAAGGCCCACCAA
GCCTTCGATCAGTAGCTGATGTGAGAGCATGATCAGCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAG
GCCTTCGATCAGTAGCTGATGTGAGAGCATGATCAGCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAG
CAGTAAGGAATATTGGTCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGAAGGATGAAGGTCCTCTGGATTGTAAAC
CAGTAAGGAATATTGGTCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGAAGGATGAAGGTCCTCTGGATTGTAAAC
TTCTTTTATAGGGGGCGAAAAAAGGGAAATCTTTCTCACTTGACAGTACCCTATGAATAAGCACCGGCTAACTCCGTGCC
TTCTTTTATAGGGGGCGAAAAAAGGGAAATCTTTCTCACTTGACAGTACCCTATGAATAAGCACCGGCTAACTCCGTGCC
AGCAGCCGCGGTAA
AGCAGCCGCGGTAA

Revision as of 09:59, 31 January 2010

16S LD-PCR 40cycle amplification for Sanger sequencing

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
Primer(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 size selected 1st PCR amplicons
11-2, 12-2: 1uL eliqote from 1st PCR amplicons

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
  • 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.

PCR product purification with Montage PCR filter

  • Mix 20uL PCR prodcut with 380uL 1XTE buffer (400uL volume in total). Mix well by vortexing 5sec.
  • Assemble the Montage filter on collecting tube.
  • Transfer the mixture onto a assembled filter.
  • Centrifuge the filter at 1000xg, for 15min at RT.
  • After centrifuge, revese the filter and put on a fresh collecting tubes carefully.
  • Centrifuge the filter at 1000xg. for 2 min to retrieve the purified PCR products.
    • The concentration purified PCR products were about ~40ng/uL in 20uL (by Nanodrop, blanked with 1X TE)

Sample dilution for Sanger sequencing at GeneWiz

  • Concentration requirement for pre-mixed sample
    • Total volume 15uL
    • PCR product: 20ng ~ 40ng in 10uL
    • Primer: 25pmole in 5uL
  • Dilution procedures
    • Step1: Mix 1uL un-diluted PCR product (~40ng/uL) with 9uL 1X TE -> 40ng of template in 10uL
    • Step2: Mix 1uL original 16S-microseq primer-f (100uM) with 19uL H2O -> 5uM, get 5uL of diluted primer (-> 25pmole)
    • Mix the solution from Step1 and step2 -> 15uL, transfer into a 8-well stripe tube. Label the sampe on the tubes.

Sequence results

  • I got very high quality score (49 and 50) for both samples.
  • For both sequence, the blast (NCBI nt database) results hit uncultureable 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