Team:Tuebingen/NotebookProtocols

From 2012.igem.org

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Revision as of 14:50, 20 September 2012



Contents

Protocols

Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

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

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

Ligation

Ligation for digested parts and vectors.

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

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

Chemotransformation

Component Volume
chemo-competent E. coli 100µl
plasmid DNA up to 10µl (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

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


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 - 4°C (program finished)

Gel electrophoresis

incl. TAE-Puffer

LB medium

incl. Agarplatten

SOB medium

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