Team:TU Darmstadt/Protocols/Metabolism

(Difference between revisions)

Protocols Metabolism

The following page give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012.

Contents 1 In vivo 1.1 Production of chemically competent cells 1.2 Heat shock transformation with E. coli 1.3 Glycerine stock 2 In vitro 2.1 PCR 2.1.1 Colony PCR (isolation of genomic DNA) 2.1.2 Colony PCR (verification of transformation) 2.1.3 PCR on a DNA template 2.2 Restriction digest

In vivo

Production of chemically competent cells

Chemically competent cells are needed for transformation with the heat shock method. We used CaCl₂ to produce them.

Production of chemically competent cells

• Inoculate 10 ml of LB-Media with an E. coli colony and incubate at 37 °C overnight
• Inoculate 200 ml of LB-Media with 2.5 ml of the overnight culture
• Incubate the culture at 37 °C until an OD600 of 0.5
• Cool the cells on ice for 5 min
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the cell-pellet in 32 ml pre-cooled CaCl₂ solution (Do not vortex!)
• Add pre-cooled CaCl₂ solution to 200 ml
• Let the cells repose on ice for 1 hour
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the pellet in 10 ml cryo-solution
• Decant 200 µl of competent cells in a 1.5 ml tube
• Store the tube in an -80 °C freezer

Solutions

• CaCl₂
  • 5.55 g CaCl₂
  • Add di H₂O to 1 L
  • Sterilize by autoclaving

• Cryo solution
  • 0.278 g CaCl₂
  • 10 ml glycerin
  • Add di H₂O to 50 ml
  • Sterilize by autoclave

Heat shock transformation with E. coli

• Thaw the chemically competent cell on ice
• Add 50 – 300 ng of the purified plasmid or 1-5 µL of the heat inactivated ligation mix to the cells (10 – 500 ng DNA)
• Invert the tube up to 6 times
• Incubate the cells on ice for 30 min
• Incubate the cells for 1 min at 42 °C
• Let the cells cool down on ice for 5 min
• Add 800 µl of SOC medium
• Incubate the cells for 45 min at 37 °C
• Centrifuge for 5 min at 0.4 rcf
• Resuspend the pellet in 100 µl LB-Media and plate it on LB Agar with antibioticum

Glycerine stock

• Add 200 µl of sterilized glycerine to 800 µl cell culture and mix well
• Freeze the stock at -20 °C

In vitro

PCR

The Polymerase Chain Reaction (PCR) is used to amplify DNA from bacterial colonies or DNA templates.

Colony PCR (isolation of genomic DNA)

• Pick one colony with a sterile tip
• One reaction mix contains:
  • 10 µL of 5x Phusion HF Buffer
  • 1 µL of dNTPs (10 mM each)
  • 0,5 µL of Phusion High-Fidelity Polymerase
  • Forward primer (10 pmol)
  • Reverse primer (10 pmol)
  • 1,5 µL of DMSO
  • DI water to 50 µL
  • Colony template

• PCR program

#	Temperature	Time
1	98 °C	00:02:00
2	Ta	00:01:00
3	72 °C	00:01:00
4	98 °C	00:01:00
5	Ta	00:01:00
6	72 °C	00:01:00
7	GO TO 4	REPEAT 31x
8	98 °C	00:01:00
9	Ta	00:01:00
10	72 °C	00:06:00
11	4 °C	HOLD

Colony PCR (verification of transformation)

• Pick one colony with a sterile tip and suspend in 10 µL of DI H₂O
• One reaction mix contains:
  • 2 µL of 10x Thermopol Reaction Buffer
  • 0,4 µL of dNTPs (10 mM each)
  • 0,3 µL of Taq DNA Polymerase
  • VF2 (10 pmol)
  • VR (10 pmol)
  • 0,6 µL of DMSO
  • 1 µL of colony suspension
  • DI water to 20 µL

• PCR program

#	Temperature	Time
1	95 °C	00:01:00
2	95 °C	00:00:20
3	62 °C	00:00:30
4	68 °C	00:02:00
5	GO TO 2	REPEAT 30x
6	68 °C	00:05:00
7	4 °C	HOLD

PCR on a DNA template

• One reaction mix contains:
  • 10 µL of 5x Phusion HF Buffer
  • 1 µL of dNTPs (10 mM each)
  • 0,5 µL of Phusion High-Fidelity Polymerase
  • Forward primer (10 pmol)
  • Reverse primer (10 pmol)
  • 1,5 µL of DMSO
  • 1 µL of template
  • DI water to 50 µL
• PCR program

#	Temperature	Time
1	98 °C	00:02:00
2	Ta	00:01:00
3	72 °C	00:01:00
4	98 °C	00:01:00
5	Ta	00:01:00
6	72 °C	00:01:00
7	GO TO 4	REPEAT 31x
8	98 °C	00:01:00
9	Ta	00:01:00
10	72 °C	00:06:00
11	4 °C	HOLD

Restriction digest

A restriction digest is used to digest either vectors or inserts via restriction enzymes before ligation.

Protocol

• One reaction mix contains:
  • DNA template (up to 3µg)
  • 2 µL NEBuffer 4 (10x)
  • 0,5 µL of restriction enzyme 1
  • 0,5 µL of restriction enzyme 2
  • 0,2 µL of 100x BSA (only when cut with SpeI-HF)
  • DI water to 20 µL
• Incubate at 37° C for 1 hour
• Heat inactivate at 80° C for 25 minutes