Team:Evry/Protocols

From 2012.igem.org

(Difference between revisions)
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<h1>Gel extraction</h1>
<h1>Gel extraction</h1>
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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.















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