Oct 09, 2023

Public workspaceOligo Pool Resuspension and PCR

DOI
dx.doi.org/10.17504/protocols.io.8epv5xe3ng1b/v1
  • 1National University of Singapore
夏雨 陈
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5xe3ng1b/v1
Protocol Citation: NUS iGEM 2023. Oligo Pool Resuspension and PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xe3ng1b/v1
Manuscript citation:
Twist Bioscience. (n.d.-a). Twist DNA Resuspension Guidelines | Twist BioScience. https://www.twistbioscience.com/resources/protocol/twist-dna-resuspension-guidelines

Twist Bioscience. (n.d.). Twist Oligo Pool Amplification Guidelines | Twist Bioscience. Retrieved October 9, 2023, from https://www.twistbioscience.com/resources/protocol/twist-oligo-pool-amplification-guidelines
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: October 08, 2023
Last Modified: October 09, 2023
Protocol Integer ID: 88977
Keywords: Oligo Pool, Resuspension, PCR, Polymerase Chain Reaction, Oligo
Abstract
2023 NUS-Singapore iGEM team followed this protocol to resuspend the dried oligo pool and perform PCR to amplify the DNA fragments in the oligo pool.
Materials
KOD OneTM PCR Master Mix (Blue)
Protocol materials
ReagentBuffer EBQiagenCatalog #19086
In 2 steps
Safety warnings
Attention
Proper lab PPE must be worn at all times.
Oligo Pool Resuspension
Oligo Pool Resuspension
Centrifuge the oligo pool for a few seconds before opening the tube.
Calculate the volume of ReagentBuffer EBQiagenCatalog #19086 required for the oligo pool (the final concentration of the sample must be at least 20ng/μL).

Add the respective volume of ReagentBuffer EBQiagenCatalog #19086 into the tube with the oligos and resuspend the dried oligos by pipetting the solution.

Oligo Pool PCR
Oligo Pool PCR
Prepare a PCR tube, and add the following reagents:
ItemVolume
KOD OneTM PCR Master Mix (Blue) 25μL
Each Primer (Two Primers In Total) 1.5μL
Oligo Pool Sample 1μL
DI Water 21μL

Run a PCR with the following setting:

TemperatureDuration
98°C 10s
60°C 5s
68°C 10s
Go to step 1, repeat the cycle 10 times
68°C 5 minutes
12°C Infinite Loop

Follow the PCR clean-up protocol to isolate the oligos from the PCR product and keep it at TemperatureRoom temperature .
Protocol
PCR Clean-up
NAME
PCR Clean-up
CREATED BY
NUS iGEM


Protocol references
Twist Bioscience. (n.d.-a). Twist DNA Resuspension Guidelines | Twist BioScience. https://www.twistbioscience.com/resources/protocol/twist-dna-resuspension-guidelines

Twist Bioscience. (n.d.-b). Twist Oligo Pool Amplification Guidelines | Twist Bioscience. Retrieved October 9, 2023, from https://www.twistbioscience.com/resources/protocol/twist-oligo-pool-amplification-guidelines