"
Team:Evry/Protocols
From 2012.igem.org
| Line 112: | Line 112: | ||
<h1>Gel extraction</h1> | <h1>Gel extraction</h1> | ||
| - | 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light. | + | 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light. <br> |
| - | 2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel. | + | 2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel. <br> |
| - | 3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved. | + | 3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved. <br> |
| - | 4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate. | + | 4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate. <br> |
| - | 5. Ass 1 gel volume of isopropanol to the sample and mix. | + | 5. Ass 1 gel volume of isopropanol to the sample and mix. <br> |
| - | 6. Place a spin column in a provided 2 ml collection tube. | + | 6. Place a spin column in a provided 2 ml collection tube. <br> |
| - | 7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube. | + | 7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube.<br> |
| - | 8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube. | + | 8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube.<br> |
| - | 9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm). | + | 9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm).<br> |
| - | 10. Place the column into a clean 1,5 ml microcentrifuge tube. | + | 10. Place the column into a clean 1,5 ml microcentrifuge tube.<br> |
| - | 11. to elute DNA, add 50 µl of water to the column and centrifugate for 1 min. | + | 11. to elute DNA, add 50 µl of water to the column and centrifugate for 1 min.<br> |
Revision as of 12:02, 7 August 2012
Contents |
PCR with Phusion High-Fidelity DNA Polymerase
Tube preparation
Put items in this order:
| Component | 50µl reaction | Comments |
| H2O | 32 | |
| 5x Phusion HF Buffer | 10 | |
| 10mM dNTPs | 1 | |
| Primer FW | 2 | Primers have to be at 10µM |
| Primer RV | 2 | Primers have to be at 10µM |
| Template DNA | 1 | |
| DMSO (optional) | 1,5 | recommended for GC-rich amplicons < 20kb |
| Phusion DNA polymerase | 0,5 |
Cycling instructions
| Cycle step | Temperature | Time | Cycles |
| Initial denaturation | 98°C | 4min | 1 |
| Denaturation | 98°C | 20s | 30 |
| Annealing | Lower Tm of primers | 30s | |
| Extension | 72°C | 30S/kb | |
| Final extension | 72°C | 10min | 1 |
| 4°C | hold |
Préparation of LB medium and LB Agar:
=> LB Agar :
-18,5g LB Agar
-300ml H2O
=> LB medium : -6g LB broth -300ml de H2O
Autoclaved at 250°C
Gel extraction
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel under UV light.
2. Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
3. Incubate at 50°C for 10 min, until the gel slice has completely dissolved.
4. The color of the mixture have to be yellow, otherwise add 10 µl of 3M sodium acetate.
5. Ass 1 gel volume of isopropanol to the sample and mix.
6. Place a spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the column and centrifugat for 1 min. Discard the flow-through and place the column back into the same tube.
8. To wash, add 0,75 ml of buffer PE to the column and centrifugate for 1 min.Discard the flow-through and place the column back into the same tube.
9. Centrifugate the column in a 2 ml Collection tube for 1 min 17,900xg (13,000 rpm).
10. Place the column into a clean 1,5 ml microcentrifuge tube.
11. to elute DNA, add 50 µl of water to the column and centrifugate for 1 min.
