Jan 17, 2019

Public workspaceSanger Sequencing

DOI
dx.doi.org/10.17504/protocols.io.pxddpi6
  • 1Stanford University School of Medicine
  • MRCA
Lívia Sacchetto
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.pxddpi6
Protocol Citation: Lívia Sacchetto, Izabela M Rezende 2019. Sanger Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.pxddpi6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 04, 2018
Last Modified: January 17, 2019
Protocol Integer ID: 11973
Abstract
For sequence by dideoxy-method using the ABI3130 platform (Applied Biosystems, USA) and specific primers.
Materials
MATERIALS
ReagentMicrocentrifuge Tubes
ReagentFilter Tips
ReagentNuclease-Free Water, 1000mlPromegaCatalog #P1199
ReagentPrimer
ReagentMicropipettors
ReagentSequencing plate
ReagentPCR purified template
ReagentSequencing buffer
ReagentSanger sequencer
Reagentadhesive plate seal
In a 0.2 mL microtube, place the items below: 
PCR product 20 to 30 ng of DNA per 100 bp
Primer Forward or Reverse 10 pmol
BigDye™ Terminator v3.1 5X Sequencing Buffer (Applied Biosystems) 2 µL
Nuclease-free waterto a final volume total of 10,0 μL
Pipette each reaction from the tube into the corresponding well of the sequencing plate (10 µL).
Seal the plate; centrifuge briefly to collect the contents of the wells at the bottom. Protect from extended light exposure or elevated temperatures before cycling.
Use Sanger sequencing with the Applied Biosystems® 3730 DNA Analyser, according to the manufacturer's instructions.