Sam:LabNotes/Microbiome-new/2010-1-28: Difference between revisions
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>Sam Chiang (Created page with '='''16S LD-PCR 40cycle amplification for Sanger sequencing'''= ==Backgorund== *I identified two possible positive amplicons by 16S-LD PCR validation. However the PCR products co…') |
>Sam Chiang |
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19.0 95.0 95/5=19 template-1uL | 19.0 95.0 95/5=19 template-1uL | ||
Template: 11-1, 11-2, 12-1, 12-2 | '''Template (1uL): 11-1, 11-2, 12-1, 12-2''' | ||
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== | |||
*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|400px]] |
Revision as of 09:21, 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