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 2.3 Ligation 2.4 Plasmid Preparation 2.4.1 Plasmid Miniprep 2.4.2 Plasmid Midiprep

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.

Protocol

• 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

The heat shock method is an effective method to transform ligation mixes or plasmids into E. coli' bacteria.

Protocol

• 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

In order to have a permanent culture of cells glycerine stocks can be made.

Protocol

• 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

Ligation

Ligation is the process of joining of DNA strands by catalyzing the formation of a phosphodiester bond. It is used to facilitate plasmid vectors.

Protocol

• One reaction mix contains:
  • 100 ng of vector DNA and x ng of insert DNA (vector:insert molar ratio is 1:5)
  • 2 µL of T4 DNA Ligase Buffer (10x)
  • T4 DNA Ligase
  • DI water to 20 µL
• Put the reaction mix into a thermocycler using the following program:

#	Temperature	Time
1	37 °C	00:00:20
2	33 °C	00:00:15
3	28 °C	00:00:15
4	23 °C	00:00:15
5	18 °C	00:00:15
6	13 °C	00:00:15
7	8 °C	00:00:15
8	4 °C	00:15:00
9	16 °C	00:12:00
10	23 °C	00:10:00
11	30 °C	00:10:00
12	37 °C	00:10:00
13	65 °C	00:15:00
14	4 °C	HOLD

Plasmid Preparation

Plasmid preparation is a method for extracting and purifying plasmid DNA from E. coli cells. The Promega PureYield^TM Miniprep and PureYield^TM Miniprep System respectively was used.

Plasmid Miniprep

Minipreps were used for yields up to 15 µg of DNA

Protocol

• Centrifuge 1.5 mL of bacterial culture at 17,000x g for 1 minute and discard the supernatant. Repeat up to a total volume of bacterial culture of 9 mL
• Follow the steps 2. - 9. of the Promega PureYield^TM Miniprep System protocol
• Place the minicolumn on a 1.5 mL tube, add 30µL of Elution Buffer and let stand at room temperature for 1 minute. Then centrifuge at 17,000x g for 1 minute
• Add 20 µL of Elution Buffer and let stand at room temperature for 1 minute. Then centrifuge at 17,000x g for 1 minute

Plasmid Midiprep

Midipreps were used for yields up to 200 µg of DNA

Protocol

• Follow the steps for elution by applying vacuum of the Promega PureYieldTM Midiprep System [http://www.promega.com/~/media/Files/Resources/ProtCards/PureYield%20Plasmid%20Midiprep%20System%20Quick%20Protocol.pdf protocol]