Amplification of DNA
Yuichiroh Ikagawa
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.
Steps
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 |
