Sam:LabNotes/Microbiome-new/2010-1-28: Difference between revisions
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>Sam Chiang |
>Sam Chiang No edit summary |
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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== | ||
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*'''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== | ||
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*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== | ||
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**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