Team:TU Darmstadt/Protocols/Metabolism

Protocols Metabolism

The following page gives an overview of the standard protocols used by the team Metabolism in the 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 Dephosphorylation 2.4 Ligation 2.5 Plasmid Preparation 2.5.1 Plasmid Miniprep 2.5.2 Plasmid Midiprep 2.6 Agarose gel electrophoresis 2.7 Gel and PCR Clean-Up 3 Protein methods 3.1 Protein overexpression 3.2 SDS-PAGE 3.3 Protein purification 4 Measurements 4.1 Nanodrop

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

• For verification of longer DNA strands, the elongation time (step 4) was prolonged. It was assumed that 1 kb/min of template was replicated by the Taq DNA Polymerase.

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

Dephosphorylation

Antarctic Phosphatase catalyzes the removal of 5' phosphate groups of DNA and RNA and thus prevents re-ligation of cut vectors. It is used before ligation.

Protocol

• The reaction mix contains:
  • up to 5 µg of digested DNA (no purification needed)
  • 1 µL of Antarctic Phosphatase
  • 1/10 of reaction volume of 10x Antarctic Phosphatase Reaction Buffer
  • 1 µL of Antarctic Phosphatase
• Incubate at 37° C for 30 minutes (3' ends) or 60 minutes (5' ends)
• Heat inactivate at 65° C for 15 minutes
• Continue with ligation

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]

Agarose gel electrophoresis

Agarose gel electrophoresis is used to separate DNA fragments of different lengths.

Protocol

• Fill 20 mL of 50x TAE buffer in 1L flask and add DI water to 1000 mL in order to have 1x TAE buffer
• Choose agarose concentration dependent on fragments size:

Agarose concentration [% (w/v)]	Fragment length [kbp]
0.8	0.5-11
1.2	0.4-7
1.5	0.2-3
2.5	0.05-2

• Measure the appropiate mass of agarose, add 1x TAE buffer to 100 mL in a flask and mix well
• Heat in a microwave at 450 W for 2-3 minutes until agarose is completely melted
• Let gel cool down to 50-60° C and pour into gel box
• Add 10 µL of Roti®-Safe GelStain and let gel harden
• Put the gel box into the gel chamber filled with 1x TAE buffer
• Mix your DNA probe with 6x Loading Dye (1 µL Loading Dye per 5 µL DNA probe)
• Remove gel comb and load one pocket with 7 µL of DNA ladder
• Load the pockets with the prepared probes
• Run the gel at 120V for about 40 minutes until the yellowish OrangeG band is close to the bottom
• Place gel in UV box and capture gel image

Media

• Loading Dye (6x)

Contents	Concentration
Glycerol	50% (v/v)
EDTA (pH 8.0)	50 mmol/L
Bromophenol blue	0.05% (w/v)
Xylene cyanol	0.05% (w/v)
Orange G	0.05% (w/v)

• TAE buffer (50x)

Contents	Concentration
TRIS	2000 mmol/L
EDTA	50 mmol/L
Sodium acetate	250 mmol/L
pH 8.0	adjust with acetic acid

Gel and PCR Clean-Up

The Wizard® SV Gel and PCR Clean-Up System was used for purifying gels and PCR products.

Protocol

• Follow steps 1. - 7. of the Wizard® SV Gel and PCR Clean-Up System [http://www.promega.com/~/media/Files/Resources/ProtCards/Wizard%20SV%20Gel%20and%20PCR%20Clean-Up%20System%20Quick%20Protocol.pdf protocol]
• Add 30 µL of Nuclease-Free Water to the minicolumn and let stand for 1 minute at room temperature. Then centrifuge at 17,000x g for 1 minute
• Add 20 µL of Nuclease-Free Water to the minicolumn and let stand for 1 minute at room temperature. Then centrifuge at 17,000x g for 1 minute

Protein methods

Protein overexpression

Protein overexpression is a subcomponent of gene overexpression and describes the massive production of a protein by an expression system, e. g. E. coli.

Protocol

• Inoculate 1 L LB-medium-antibiotica with 3 ml bacteria suspension (OD₆₀₀ = 1.0) and incubate over night
• Measure the OD₆₀₀, take 1 mL of the culture and centrifuge the 1 mL sample at 17,000x g for 1 min
• Calculate the amount of diH₂O and Hoechst buffer added to the sample
  • Example: For an OD₆₀₀ = 1 you have to add 100 µl diH₂O and 50 µl Hoechst buffer, for an OD₆₀₀ = 2 you have to add 200 µl diH₂O and 100 µl Hoechst buffer and so on
• After centrifugation discard the supernatant and add the diH₂O and Hoechst buffer
• Freeze the sample at -20 °C
• Induce with 150 µl of 1 M IPTG Solution
• Measure hourly the OD₆₀₀, take 1 mL of the culture and centrifuge the 1 mL sample at 17,000x g for 1 min
• After centrifugation discard the supernatant and add the diH₂O and Hoechst buffer
• Freeze the sample at -20 °C
• After 4 hours centrifuge the over expression culture at 4,600x g and resuspend the pellet with the wash buffer from protein purification
• Storage the suspension at -20 °C or continue with protein purification
• Perform an SDS-PAGE with the frozen samples

SDS-PAGE

Protein purification

Measurements

Nanodrop

DNA absorbs light at 260 nm and thus the concentration can be measured photometrically using the [http://www.nanodrop.com/Library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop 1000 Spectrophotometer].

Protocol

• Start the programme for the NanoDrop 1000 and click “Nucleic Acids”
• Pipette 1.5 µL of a water sample onto the lower measurement and click “OK”
• Load your blank and click “Blank”
• Load your DNA samples and click “Measure”. A 260/280 ration of 1.8 or above means the sample can be seen as pure
• At the end load your blank and click “Measure” to test the blank. If the measurement deviates more than 5 ng/µL from 0.0 ng/µL, load again the blank, click “Blank” and measure your DNA samples again