Team:Tuebingen/NotebookProtocols

From 2012.igem.org

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= Protocols =
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== Protocols ==
 
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=== Chemo-competent cells ===
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== Chemo-competent cells ==
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=== pGEM Ligation ===
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== pGEM Ligation ==
Ligation for TA-cloning of PCR products
Ligation for TA-cloning of PCR products
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Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
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=== Ligation ===
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== Ligation ==
Ligation for digested parts and vectors
Ligation for digested parts and vectors
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Mix all reagents and incubate at 22°C for 1 hour.
Mix all reagents and incubate at 22°C for 1 hour.
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=== Chemotransformation ===
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== Chemotransformation ==
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# Let the bacteria grow at 37°C at least for 1 hour.
# Let the bacteria grow at 37°C at least for 1 hour.
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=== Restriction digest ===
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== Restriction digest ==
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==== control digest ====
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=== control digest ===
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==== preperative double digest ====
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=== preperative double digest ===
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==== plasmid linearization ====
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=== plasmid linearization ===
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=== PCR ===
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== PCR ==
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=== Gel electrophoresis ===
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== Gel electrophoresis ==
incl. TAE-Puffer
incl. TAE-Puffer
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=== LB medium ===
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== LB medium ==
incl. Agarplatten
incl. Agarplatten
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=== SOB medium ===
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== SOB medium ==

Revision as of 15:18, 22 September 2012



Protocols

Contents


Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

Component Volume
2X Rapid Ligation Buffer 5 µl
pGEM vector 0.5 µl (25ng)
PCR product 3.5 µl
T4 DNA ligase 1 µl (3 Weiss units)

Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.

Ligation

Ligation for digested parts and vectors

Component Volume
10X T4 DNA Ligase Buffer 1 µl
vector DNA 1 µl (20-100 ng)
insert DNA 5 µl (up to 5:1 molar ratio insert to vector)
T4 DNA ligase 1 µl (1 unit)
water 2.5 µl

Mix all reagents and incubate at 22°C for 1 hour.

Chemotransformation

Component Volume
chemo-competent E. coli 100 µl
plasmid DNA up to 10 µl (max. 1/10 of volume)
  1. Add plasmid DNA to cell culture.
  2. Incubate for 30 min on ice.
  3. Heat shock for 90 sec at 42°C.
  4. Add 900 µl LB.
  5. Let the bacteria grow at 37°C at least for 1 hour.

Restriction digest

control digest

preperative double digest

plasmid linearization

PCR

Component Volume
Taq/Pfu buffer 5 µl
Taq/Pfu polymerase 1 µl
primer forward 0.5 µl (100 pmol/µl)
primer reverse 0.5 µl (100 pmol/µl)
dNTPs 2.5 µl (200 µM)
template DNA 1 µl
water 36 µl

PCR conditions

Step Duration Settings
1 2 min 94°C
2 45 sec 94°C
3 30 sec gradient or annealing temperature
4 90 sec 72°C
steps 2-4: 30 cycles
5 7 min 72°C
6 (hold) 4°C

Gel electrophoresis

incl. TAE-Puffer

LB medium

incl. Agarplatten

SOB medium

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