Sam:LabNotes/Microbiome-new/2010-1-28: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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**Ladder: 5uL pre-diluted 1kb-plus ladder | **Ladder: 5uL pre-diluted 1kb-plus ladder | ||
[[File:sam012810-2nd PCR.jpg| | [[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. | |||
==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''' | |||
**PCR product: |
Revision as of 09:35, 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
- PCR product: