Nov 25, 2024
Sanger Sequencing Preparation and Submission
This protocol is a draft, published without a DOI.
- Raven Lewis1
- 1Purdue University
Protocol Citation: Raven Lewis 2024. Sanger Sequencing Preparation and Submission. Protocol exchange https://protocols.io/view/sanger-sequencing-preparation-and-submission-drn455gw
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: November 13, 2024
Last Modified: November 25, 2024
Protocol Integer ID: 112060
Keywords: sanger sequencing, picogreen, pcr
Abstract
This protocol describes the preparation and submission process for Sanger sequencing. Specifically, this protocol discusses the preparation, process, and clean-up for PCR, as well as the way to submit samples for Sanger sequencing.
As a result of following this protocol, it is expected that 50 µL of purified PCR product will be obtained for submission for Sanger sequencing. At the end of this protocol, an aliquot of these purified products will be submitted for Sanger sequencing.
Materials
PicoGreen Dye Reagent
PicoGreen Kit TAE
Kit example: Quant-iT™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P11496
Molecular Grade Water
Q5 high-fidelity master mixNew England Biolabs
Bacterial Isolates
Previously isolated genomic DNA
Target primers
2 mL Microcentrifuge tube(s)
200 µL PCR tubes
96-Well PCR plate(s)
Aluminum foil
Pipettes and Tips
Inoculation loop(s)
Vortex Mixer
Thermocycler
Microcentrifuge
Plate Spinner
qPCR Instrument
-20 °C freezer
QIAquick PCR Purification KitQiagenCatalog #28104
Protocol materials
Q5 high-fidelity master mixNew England Biolabs
Materials, Step 3
QIAquick PCR Purification KitQiagenCatalog #28104
Materials
Quant-iT™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P11496
Materials
Safety warnings
The PicoGreen used in this protocol is photosensitive. Ensure that it is always wrapped in aluminum foil, and try to minimize the amount of time it is in the presence of light.
Before start
Ensure that you have all the required materials to successfully complete this protocol. See the materials section for a list of what is required.
Create PicoGreen Working Stock
Create PicoGreen Working Stock
Thaw the PicoGreen dye reagent at room temperature in a place protected from light.
Note
Ensure that the PicoGreen dye reagent is wrapped in aluminum foil, as it is photosensitive.
Prepare 4X PicoGreen (dsDNA quant solution).
Label a 2 mL microcentrifuge tube with "4X Pico". Wrap this microcentrifuge tube in aluminum foil.
Prepare 0.5 µL of 1X TAE buffer and molecular-grade water. To do this, add 25 µL of PicoGreen kit TAE (20X) and 475 µL of molecular-grade water to the labeled microcentrifuge tube.
Add 10 µL of fully thawed PicoGreen dye to the labeled microcentrifuge tube.
Note
This is not exactly 4X, but it does not matter.
Prepare PCR Master Mix
Prepare PCR Master Mix
Add 40 µL of your 4X Pico to one whole tube of Q5 high-fidelity master mixNew England Biolabs (1250 mL ).
Boiling Lysis DNA Extraction and PCR for Sanger Sequencing
Boiling Lysis DNA Extraction and PCR for Sanger Sequencing
For each isolate, label:
1) 200 µL PCR tube for initial lysis (Tube Set 1)
2) 200 µL PCR tube for dilution (Tube Set 2)
Additionally, obtain and label the required amount of 96-well PCR plates. The amount will vary based on the number of your samples.
Note
Include tubes for a positive control (previously isolated genomic DNA) and a negative control. You do not need to label a lysis tube for the positive control.
Add 125 µL of molecular-grade water to Tube Set 1 for boiling lysis.
Add culture.
If using a solid colony, add a loopful of colonies into the water. Use a sterile inoculating loop for the negative control.
If using a liquid culture, add 50 µL of the vortexed culture to the water. For the negative control, add 50 µL of molecular-grade water.
Vortex to mix.
Load Tube Set 1 into a thermocycler. Use the parameters:
1) 99 °C for 00:15:00
2) 4 °C for temporary storage
Move to a -20 °C freezer for 00:05:00 .
8000 rpm, 00:05:00 .
Dilute newly obtained lysate in Tube Set 2.
Add 18 µL of molecular-grade water.
Add2 µL of your spun-down sample.
Note
Be careful to avoid disturbing the cell pellet.
Add 2 µL of previously isolated genomic DNA for the positive control.
To prepare PCR reactions, use the attached spreadsheet. Add the calculated quantities to your 96-well plate(s).
Note
This spreadsheet uses the primer pair 27F-1492R.
Spin down PCR samples in a plate spinner.
Load into qPCR machine. Use the program:
1) 98 °C for 00:03:00
2) 30 cycles:
1. 94 °C for 00:01:00
2. 55 °C for 00:01:00
3. 72 °C for 00:00:45
3) 72 °C for 00:15:00
4) 4 °C for temporary storage
PCR Product Purification
PCR Product Purification
Follow the attached protocol from Qiagen.
Submit Sanger Sequences
Submit Sanger Sequences
Follow the steps listed for sample submission.