Oct 21, 2021
Amplification of DNA
- 1iGEM Gifu
- iGEM Gifu
Protocol Citation: Yuichiroh Ikagawa 2021. Amplification of DNA . protocols.io https://dx.doi.org/10.17504/protocols.io.bzbcp2iw
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: In development
We are still developing and optimizing this protocol
Created: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54340
Abstract
Our project aims to quantify human fatigue through the quantification of the biomarker human herpesvirus6 using Cas12a, a protein that is fundamental and essential to our project. We ordered the synthesis of composite parts with affinity tags attached to Cas12a, but the sequence was too long to be synthesized as a single of dsDNA. To overcome this problem, we split the parts into three fragments and synthesized them. Here, an adaptor sequence was added to the 5' end by PCR to allow for later assembly.
Materials
Reagent
KAPA HiFi HotStart ReadyMix (x2) (KAPA Biosystems)
Vector primers (IDT) :
- Forward
5'- CCATACTACGTTTGAGGAGGATATCGTTGCCTGAGCGAAGGTCCGGCAAAAAAGGGCAAG -3'
- Reverse
5'- GACAGAGCTCTACGGACTGTAACGCGTTTGGTACTGCAATCCAGAAATCATCCTTAGCG -3'
Fragment primers:
- Fragment 1 Forward
5'- ATTGCAGTACCAAACGCGTTACAGTCCGTAGAGCTCTGTC -3'
- Fragment 1 Reverse
5'- CTCAGGCAGGATGGTTTCAATAATGTCTTTTTTAAACAGAG -3'
- Fragment 2 Forward
5'- TCTGTTTAAAAAAGACATTATTGAAACCATCCTGCCTGAG -3'
- Fragment 2 Reverse
5'- AGTACATGGTATGTAAGTTCGGCGTGCCATGGCTCTTGTC -3'
- Fragment 3 Forward
5'- GACAAGAGCCATGGCACGCCGAACTTACATACCATGTAC -3'
- Fragment 3 Reverse
5'- CTTCGCTCAGGCAACGATATCCTCCTCAAACGTAGTATGG -3'
Template DNA:
- pSB1C3
- pSB1A3
- Cas12a fragment 1 (Twist Bioscience)
- Cas12a fragment 2 (Twist Bioscience)
- Cas12a fragment 3 (Twist Bioscience)
DNase free water
Equipment
Thermal cycler:
- Gene Amp® PCR system 2700 (Applied Biosystems)
- LifeECO ver3.0 (Bioer Technology)
Thaw the DNA solution at room temperature, and KAPA HiFi HotStart ReadyMix (x2) on ice.
Vortex the reagents, then centrifuge them briefly in a microcentrifuge.
Mix the reagents according to the composition in the table below.
| A | B | C | |
| COMPONENT | VOLUME (µl) | CONCENTRATION | |
| KAPA HiFi HotStart Ready Mix(x2) | 25 | ×2 | |
| DNA Template | 1.0 | 1.0 µg/µl | |
| Forward Primer | 2.5 | 0.5 µM | |
| Reverse Primer | 2.5 | 0.5 µM | |
| Nuclease Free Water | 19.0 | ||
| Total Volume | 50.0 |
For each template DNA, select the temperature setting of the thermal cycler listed in the table below.
Vectors(pSB1C3 and pSB1A3)
| A | B | C | D | |
| STEP | TEMP (℃) | TIME | CYCLES | |
| Initial Denature | 95 | 5 minutes | ||
| Denature | 98 | 30 seconds | 30 | |
| Anealing | 68 | 15 seconds | ||
| Extension | 72 | 1 minutes 30 seconds | ||
| Final Extension | 72 | 5 minutes | ||
| Hold | 4 |
Cas12a fragments(1-3)
| A | B | C | D | |
| STEP | TEMP(℃) | TIME | CYCLES | |
| Initial Denature | 95 | 5 minutes | ||
| Denature | 98 | 30 seconds | 30 | |
| Anealing | 65 | 15 seconds | ||
| Extension | 72 | 1 minute | ||
| Final Extension | 72 | 5 minutes | ||
| Hold | 4 |