IGEM:IMPERIAL/2007/Notebook/General Protocols

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Transformation

Summary: inserting plasmids into bacterial cells

Preparation of Competent Cells

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1. Transferred 25ml of fresh overnight culture to 1 litre of LB
2. Grew cells with vigorous aeration (at least 225 rpm) to OD600 of 0.5-0.6 (1.6-1.9 x 10^8 cells/ml)
3. Transferred culture to sterile 500 ml centrifuge bottles
4. Allowed to cool on ice for several minutes
5. Pellet cells for 15 min at 4000 x g
6. Resuspended cells in 1 litre of ice cold ddH20
7. Repelleted cells immediately
8. Repeated steps 6 and 7
9. Set up 0.5 ml eppendorf tubes in rack on dry ice bath to chill
10. Resuspended cells in 20 ml 10% glycerol
11. Transferred to 50 ml centrifuge tube
12. Pelleted cells for 15 min at 4000 x g
13. Resuspended cells in 2 ml of 10% glycerol
14. Prepared 50 μl aliquots
15. Froze on dry ice
16. Stored at -70°C
17. Plated dilutions of the final preparation

Electroporation

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1. Take out E. coli DH5-α from -80°C and thaw on ice
2. Place cuvettes on ice
3. Switch on the electroporation machine. Set to voltage to 1.67
4. Take 1μL of DNA and add it into the 50μL of competant E. coli, mix carefully and transfer to cuvette
   
5. Set up the cuvette on the electroporation machine and zap!
   [Note: If you hear a pop (the read is not around ~4.30), then repeat the experiment for that plasmid]
6. Remove the zapped cells from the cuvette and add 0.5 ml LB
7. Transfer back to the eppendorf and grow for 1 hour in a shaking incubator at 37°C
8. Pellet down the cells and concentrate to approximately 50 μl
9. Plate the 5 μl and 50 μl onto the correct resistance plate
10. Incubate in 37°C overnight

Plasmid Preparation

Summary: extracting plasmid DNA from bacterial cells

Miniprep

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1. Transfer 1.5 ml of bacterial cells to an eppendorf
2. Pellet cells by centrifuging for 30 seconds at maximum speed
3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to an eppendorf
4. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
5. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
6. Centrifuge for 10 min at maximum speed
7. Pipette the supernatant into the QIAprep spin column on top of the eppendorf
8. Centrifuge for 1 min and discard the flow-through.
9. Wash with 0.5 ml Buffer PB, centrifuge for 1 min, and discard the flow-through
   [Note: This step is unnecessary for cells without nucleases (DH5α, XL1-Blue]
10. Wash with 0.75 ml Buffer PE
11. Centrifuging for 1 min and discard the flow-through
12. Centrifuge for an additional 1 min to remove residual wash buffer
13. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube
14. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min

Midiprep

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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5ml LB medium containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorous shaking (~300 rpm)
2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 50ml medium. For low-copy plasmids, inoculate 150ml medium. Grow at 37°C for 12-16h with vigorous shaking (~300 rpm)
3. Harvest the bacterial cells by centrifugation at 60000 x g for 15 min at 4°C
4. Resuspend the bacterial pellet in 6ml buffer P1
5. Add 6ml buffer P2, mix thoroghly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C)for 5 min
6. Add 6ml chilled buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Do not incubate the lysate on ice
7. Pour the lysate into the barrel of the QIAfilter Catridge. Incubate at room temperature for 10 min. Do not insert the plunger
8. Equilibrate a HiSpeed Midi Tip by applying 4ml buffer QBT and alow the column to empty by gravity flow
9. Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi catridge and filter the cell lysate into the previously equilibrated HiSpeed Tip
10. Allow the cleared lysate to enter the resin by gravity flow
11. Wash the HiSpeed Midi Tip with 20ml buffer QC
12. Elute DNA with 5ml buffer QF
13. Precipitate DNA by adding 3.5ml room temperature isopropanol to the eluted DNA. Mix and incubate at room temperature for 5 min
14. During the incubation remove the plunger from a 20ml syringe and attach the QIAprecipitator Midi Module onto the outlet nozzle. Do not use excessive force, bending, or twisting to attach the QIAprecipitator
15. Place the QIAprecipitator over a waste bottle, transfer the elute/isopropanol mixture into the 20ml syringe, and insert the plunger. Filter the elute/isopropanol mixture through the QIAprecipitator using constant pressure
16. Remove the QIAprecipitator from the 30ml syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2ml 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through QIAprecipitator using constant pressure
17. Remove the QIAprecipitator from the 20ml syringe and pull out the plunger. Attach the QIAprecipitator to the 20ml syringe again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitator quickly and forcefully. Repeat this step
18. Dry the outlet nozzle of the QIAprecipitator with absorbent paper to prevent ethanol carryover
19. Remove the plunger from a new 5ml syringe and attach the QIAprecipitator onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a 1.5ml collection tube. Add 1ml of buffer TE to the 5ml syringe. Insert the plunger and elute the DNA into the collection tube using constant pressure
20. Remove the QIAprecipitator from the 5ml syringe, pull out the plunger and reattach the QIAprecipitator to the 5ml syringe
21. Transfer the elute from step 19 to the 5ml syringe and elute for a second time into the same 1.5ml tube

Preparation of Media

Summary: preparing nutrient broths where bacteria grow

LB Medium

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To make 1 L of LB (Luria-Bertani) medium:

• 1 L distilled water
• 10 g tryptone
• 5 g yeast extract
• 5 g NaCl
• 1 ml 1 M NaOH

1. Autoclave for 25 minutes and store at room temperature

[Note: Please minimize contamination to the medium during preparation and after use. If LB us suspected to be contaminated, make a new one.]

LB Plates

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To make 1 L of LB medium for 25-30 plates:

• 10 g tryptone
• 5 g yeast extract
• 5 g NaCl
• 1 ml 1 M NaOH
• 15 g agar or agarose

1. Autoclave for 25 minutes and wait until hand-hot (50°C)
   [Note: Make sure the agar does not cool too much and solidify]
2. Add:
   • Ampicillin to 50 µg/ml
   • Kanamycin to 30 µg/ml
   • Xgal to 20 µg/ml
   • IPTG to 0.1 mM
3. Pour in 25-30 plates
4. Cool at room temperature for 2-3 days or with lids open for 30 min in 37°C incubator with laminar flow hood
5. Wrap and store at 4°C
   [Note: Please label the plates with the correct colour of antibiotics]

SB Medium

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To make 1 L of SB (super broth) medium:

• 32 g tryptone
• 20 g yeast extract
• 5 g NaCl
• 5 ml 1 M NaOH

2x YT Medium

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To make 1 L of 2x YT (TY, trypton-yeast) medium:

• 16 g tryptone
• 10 g yeast extract
• 5 g NaCl

M9 Medium

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Detail protocols of M9 Medium can be found in Cell By Date protocols

Antibiotic Stocks

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• 50 mg/ml Ampicillin
• 30 mg/ml Kanamycin

Preparation of Stock Solutions

Summary: preparing buffers

Tris-Acetate

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To make 1 L of 50x Tris-acetate solution:

• 242 g Tris base
• 57.1 ml glacial acetic acid

1. Add H20 to 1 L

Glucose/Tris/EDTA Solution

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• 50 mM glucose
• 25 mM Tris·Cl, pH 8.0
• 10 mM EDTA

1. Autoclave and store at 4°C

NaOH/SDS Solution

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[Note: Prepare NaOH/SDS solution immediately before use]

• 0.2 M NaOH
• 1% (wt/vol) sodium dodecyl sulfate (SDS)

Potassium Acetate Solution

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5 M potassium acetate solution, pH 4.8

• 29.5 ml glacial acetic acid

1. Add KOH pellets until solution reaches pH 4.8
2. Add H₂O to 100 ml
3. Store at room temperature (do not autoclave)

Restriction Digest

Summary: using enzymes to cut DNA on specific sites - this is used to cut at the suffix or prefix of the biobrick plasmids

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In biobricks: EcoRI and XBaI are in the prefix and SpeI and PstI are in the postfix.
[EcoRI - XBaI - GENE - SpeI - PstI]
In host plasmid (the plasmid you keep):

• Insert at prefix: EcoRI, XBaI
• Insert at postfix: SpeI, PstI

In donor plasmid (the plasmid you don't want):

• Insert to prefix: EcoRI, SpeI
• Insert to postfix: XBaI, PstI

For a 20 µl double restriction enzyme digest

• x μl DNA (0.1 to 4 μg DNA)
• 2 μl 10x restriction buffer
• 2 μl of restriction endonuclease/s (1 to 5 U/μg DNA)
• Add up H₂O to a total volumne of 20 μl

1. Incubate the reaction mixture for 1 hr at about 37°C
   [Note: Digest the DNA with the enzyme(s)active at the lower NaCl concentration]
2. Stop the reaction - two ways to do so:
   • EDTA (should not be used if DNA is to be used in subsequent enzymatic reactions)
   • Incubating 10 min at 65°C (some enzymes may be completely or partially resistant - inactivated by incubating 15 min at 75°C)

Solutions Required

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Tris/EDTA Buffer

• 10 mM Tris×Cl
• 1 mM EDTA, pH 8.0

Tris×Cl

• Tris×Cl [tris(hydroxymethyl)aminomethane], 1 M

1. Dissolve 121 g Tris base in 800 ml H₂O
2. Adjust to desired pH with concentrated HCl
3. Mix and add H₂O to 1 liter

Ligation

Summary: sticking the DNA together

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Set up the following ligation reaction:

• 9 μl component DNAs (0.1 to 5 μg)
• 10 μl 2× ligase buffer
• 1 μl 10 mM ATP
• 20 to 500 U (cohesive end) T4 DNA ligase

1. Incubate 1 to 24 hr at 15°C.

Agarose Electrophoresis

Summary: method to seperate DNA of different sizes, using an electric field, in a gel

Preparing the gel

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1. Seal the gel casting platform with tape and insert the gel comb
2. Measure out ~ 1.2% agarose (1.2 g of agarose for every 100 ml of TBE)
3. Mix with appropriate amount of diluted TBE suitable for the gel platform
4. Melt the agarose a microwave oven (1-2 min) and swirl to ensure even mixing
5. Melted agarose must be cooled to 55°C before pouring onto the gel platform
6. Add the dye to visualise DNA
7. Pour in the melted agarose
   [Note: Make sure there are no air bubbles on the gel, remove any with a pipette tip]
8. Allow 30-60 minutes for the gel to set

Loading and running the gel

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1. Remove the tape from the open ends of the gel platform and withdraw the gel comb
2. Place the gel casting platform containing the set gel in the electrophoresis tank
3. Add sufficient TBE to cover the gel, until the tops of the wells are submerged.
   [Note: Make sure no air pockets are trapped within the wells]
4. Load the DNA into the wells, with a suitable weight marker (make sure DNA is mixed with loading buffers)
5. Set the voltage to the desired level (100-150 V)
   The progress of the separation can be monitored by the migration of the dyes in the loading buffer
6. Turn off the power supply when the dye just before the dye reaches the end of the gel
7. Take a picture of the gel

Solutions Required

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Tris-Borate-EDTA

To make 10x TBE:

• 108g Tris
• 55g boric acid
• 7.44g di-sodium EDTA
• Make up to 1 litre with ddH₂O

DNA Extraction/Purification

Summary: extracting the DNA from the gel

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See instructions on kit