From 2011.igem.org

Latest revision as of 03:16, 29 September 2011

ITESM MÉXICO

SensE.coli

Protocols

E. coli Calcium Chloride competent cell protocol

• Materials

Equipment

• Centrifuge
• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• 0.1M CaCl2
• Glycerol

1. Inoculate a single colony (diameter: 2-3mm) into 100mL Lb in a falcon tube. Shake @ 37°C for 3hrs, 150-200rpm

a.From an ON culture (single colony into 5 mL LB in 50 mL falcon 37°C, 250 rpm), inoculate 1 mL into 100 mL LB (0.25 mL from ON culture into 25 mL LB in 50 mL falcon)

1. When the O.D. 600=0.35 put the cells on new tubes on ice for 10-15 mins (keep cold form now on).
2. Collect the cells by centrifugation 2700g (4100rpm) for 3 min at 4ºC
3. Decant supernatant (be careful not to dump out pellet, and drain tube on paper towel).
4. Gently resuspend on 10 mL cold 0.1M CaCl2 (cells are susceptible to mechanical disruption, so treat them nicely).
5. Incubate on ice for 15 min
6. Repeat 3 and 4.
7. Gently resuspend pellet on 2mL cold 0.1MCaCl2/15%Glycerol (2ml 0.1MCaCl2/50ml bacterial culture)
8. Dispense in microtubes. Freeze in -80°C.

REHYDRATION

• Materials

Equipment

• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• Distilled water

Biological agents

• Resuspended DNA. DNA kit plate

To use the DNA in the Distribution Kit you may follow these instructions:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells. But it is important to take care of the plate orientation after punching the foil.
2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 2µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.

TRANSFORMATION

• Materials

Equipment

• Incubator
• Water bath
• Petri dishes (with LB agar and appropriate antibiotic/ two per a transformation)
• Pipette tips
• Micropipettes
• Sterile tubes
• Sterile loops
• Racks

Reagents

• LB agar

• LB broth
• Distilled water
• Antibiotics (according to each BioBrick)

Biological agents

• Resuspended DNA
• Competent cells

1. Start thawing the competent cells on crushed ice.
2. Add 100 µL of thawed competent cells and then 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
3. Incubate the cells on ice for 30 minutes.
4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 2min. A water bath improves heat transfer to the cells.
5. Incubate the cells on ice for 5 minutes.
6. Add 200 μl of LB broth (make sure that the broth does not contain antibiotics and is not contaminated)
7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
9. Incubate the plate at 37ºC for 16 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.
10. Pick a single colony and inoculate broth (with the correct antibiotic) and grow for 16 hours.
11. Use the resulting culture to miniprep.

Glycerol stock

• Materials

Equipment

• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• 60ml glycerol: 4ml CaCl2: 36 ml de H2O
• 1ml glycerol/ 1 ml bacteria

1. Add 1 ml of 60% glycerol in H2O to a falcon tube.
2. Add 1 ml sample from the culture of bacteria to be stored.
3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.

a. Alternatively, pipet to mix.

1. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
2. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
3. Store in a freezer box in a -80ºC freezer. Remember to record where the vial is stored for fast retrieval later.

Miniprep Plasmid DNA Isolation

• Materials

Equipment

• Centrifuge
• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• Solutions:

• Solution 1:

• 1.8g→ 50 mM glucose
• 0.6057g→ 25 mM Tris-HCl pH 8.0:
• 0.744g→ 10 mM EDTA pH 8.0:
• Add H2O to 200 ml.

• Solution 2:

• 2g→ 1% SDS. Add H2O to 198 ml.
• Add 1.6g→ 0.2 N NaOH
• Add H2O to 200 ml.

• Solution 3:

• 98.14g→ 5 M Potassium Acetate
• Add glacial acetic acid to 200 ml.

• TE:

• 0.2428g → 10 mM Tris-HCl pH 8.0
• 0.744g→ 1 mM EDTA
• Add H2O to 200 ml.

• Optional: RNAse can be added to TE at final concentration of 20 µg/ml.

Biological agents

Bacterial culture grown

1. Fill a centrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in centrifuge for 1 minute, and make sure tubes are balanced in centrifuge. Dump supernatant and drain tube briefly on paper towel.
2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel. If necessary, repeat this step (depending on the amount of pellet)
3. Add 2 ml ice-cold Solution 1 (GTE) to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

a.Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH (8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.

1. Add 4 ml Solution 2 (NAOH + SDS 1%), cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.
2. Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.
3. Add 3 ml ice-cold Solution 3 (Potassium acetate), cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.

a.Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.

1. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh centrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
2. This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
3. Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes.
4. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
5. Centrifuge tubes for 10 minutes (13.4krpm max). A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
6. This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
7. Add 10 ml of ice-cold absolute ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
8. Ethanol helps to remove the remaining salts and SDS from the preparation.
9. Allow tube to dry for ~5 minutes. Add 500 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer.

Preparing Plates, Solutions, and Bacterial Starter Plate

• Follow the kit directions to prepare and pour the agar plates, and to rehydrate the provided lyophilized materials such as E. coli bacteria, antibiotics, DNA, etc.

1. Prepare and pour the agar plates—LB only (LB) and LB plus ampicillin (LB:AMP).

a. Label the plates with permanent marker: LB and LB:AMP.

b. After the agar solidifies, cover the plates, put them in their original plastic bags, and store them in a lab refrigerator stacked upside down. Store plates wrapped up in their original plastic wrappings. Storing upside down will ensure condensation does not wet the surface of the agar.

c. Note that the pGlo Transformation kit also allows for visualization of the transformation. In addition to the acquisition of Ampicillin resistance, the transformed bacteria can also express another gene on the pGLO plasmid which causes the bacteria to glow a brilliant green color. In order to see this, prepare the LB:AMP:ARA agar plates as specified in the pGLO transformation kit product insert. After transformation on day 2, plate the transformed cells on the LB:AMP:ARA plates as well. The arabinose in the agar will induce expression of the green fluorescent protein and the bacteria will glow green. While this step is cool to see it is not required for you to determine transformation efficiency. The Bio-Rad pGLO transformation kit comes with one UV penlight. This should be sufficient to visualize the glowing bacteria. However if you have access to a laboratory with a long wave UV lamp, that will be great. Caution - Do not shine UV light directly into the eyes, use a UV-protective face shield or goggles, and limit exposure to UV light.

1. Rehydrate bacteria and streak LB starter plates.
2. Incubate starter plates overnight at 37°C (or 2 to 3 days at room temperature until colonies are clearly visible). 5 mL Overnight

What you will need:

• 10 mL culture tube Use 16mm x 160mm or 16mm x 125 mm
• 5 mL LB
• 5uL 1000X antibiotics
• Single colonies on a plate Best not to start O/N from glycerol stocks

Procedure

1. Pipet 5uL 1000X antibiotic into culture tube.

1. Add 5 mL non-contaminated LB. Do this first, then add antibiotic
2. Select single colony using sterile toothpick or a flamed loop that has been cooled
3. Place toothpick or loop in culture tube, stir.
4. Remove toothpick or loop and place culture tube in incubator at 37 C overnight shaking vigorously (250 rpm).

Annexes

SOC media preparation

Materials

• SOB media
• 20 mM glucose

Protocol

1. Follow directions to make 1 liter of SOB media
2. After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution

SOB media preparation

Materials

• 0.5% (w/v) yeast extract
• 2% (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO4

Per liter:

• 5 g yeast extract
• 20 g tryptone
• 0.584 g NaCl
• 0.186 g KCl
• 2.4 g MgSO4

Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4. SOB medium is also available dry premixed from Difco, 0443-17. Important: Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

EDTA preparation

• 0.5 M EDTA stock
• 18.61 g EDTA (Sodium Salt)
• dH2O to 90 ml
• adjust pH to 7.0
• adjust volume to 100 ml

Agarose Gel Electrophoresis

• Material:

• 1X TAE
• Graduated cylinder
• 125 mL flask
• Agarose
• Gel pouring tray
• Tape
• Gel rig
• Ethidium bromide

Procedure:

1. Dilute stock of 10X TAE to 1X with ddH2O.
2. Measure 40 mL of buffer.
3. Transfer buffer to 125 mL flask
4. Weigh out enough agarose to make 1% gel. (1% of 40mL is 0.40 g)
5. Transfer agarose to 125 mL flask.
6. Melt agarose in microwave, stirring ever 15-20 seconds. This should take about 2 min.
7. Allow agarose to cool.
8. While agarose is cooling, assemble gel pouring apparatus by inserting gate into slots.
9. Use a pastuer pipet to run a bead of molton agarose along the edges of the gates to seal the box and prevent leaks.
10. Allow gel to cool until flask can be handled comfortably.
11. Place comb in the gel rig.
12. Pour agarose into gel tray.
13. Allow to solidify. While the gel is solidifying, prepare the samples. Add your sample and 2uL of OG loading dye to a tube, then make the total volume of the tube up to 20 uL.
14. Pour 1X TBE over gel so that gel is covered by 3-5 mm of buffer.
15. Load samples into lane. Do not forget to load 1kb+ ladder into one of the lanes.
16. Hook electrodes to gel apparatus. Nucleic acids are negatively charged, so they will run to the positive (red) terminal.
17. Pipette 10 uL ethidium bromide into the buffer at the bottom of the gel. Mix well
18. Turn on the gel. Run for 60 min @ 90V. Check with handheld UV Source.
19. Place gel in plastic wrap.
20. Carry to g311.
21. With bare hands log in as Gen 420 with password Molecular1.
22. Double click on the Genesnap from Syngene icon.
23. Click on the Green Button to start live image.
24. Put one glove on your left hand and place gel on transilluminator. Now do not touch anything with your left hand.
25. With your right hand slide the door down completely.
26. The transilluminator image on the screen should turn purple.
27. Use the arrows on the exposure button to increase the exposure time until the gel and bands are clearly visible.

Butanerds Protocols 8

1. If necessary use the zoom arrows to increase or decrease the size of the gel.
2. Reposition the gel if necessary – open the door with your right hand and move the gel with your gloved left hand.
3. To fine focus the image use the eye arrows.
4. When the image is sized and focused properly capture it by clicking the red button.
5. Print a photograph by clicking the printer icon in the tool bar at the top.
6. Record your use on the sheet. Supervisor=iGEM and your initials.
7. Log off.
8. Remove your gel and clean the transilluminator with water and dry with paper towels.
9. Take the gel back to the lab.

Biological agents

• Bacterial culture grown

1. Fill a centrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in centrifuge for 1 minute, and make sure tubes are balanced in centrifuge. Dump supernatant and drain tube briefly on paper towel.
2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel. If necessary, repeat this step (depending on the amount of pellet)
3. Add 2 ml ice-cold Solution 1 (GTE) to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

a. Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH (8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.

1. Add 4 ml Solution 2 (NAOH + SDS 1%), cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.

a. Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.

1. Add 3 ml ice-cold Solution 3 (Potassium acetate), cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.

a. Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.

1. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh centrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
2. This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
3. Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes
4. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
5. Centrifuge tubes for 10 minutes (13.4krpm max). A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
6. This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
7. Add 10 ml of ice-cold absolute ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
8. Ethanol helps to remove the remaining salts and SDS from the preparation.
9. Allow tube to dry for ~5 minutes. Add 500 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer.

Backbone protocol

• Digestion Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns)

• 5 ul NEB Buffer 2
• 0.5 ul BSA
• 0.5 ul EcoRI-HF
• 0.5 ul PstI
• 0.5 ul DpnI (Used to digest any template DNA from production)
• 18 ul dH20

Procedure:

• Digest Plasmid Backbone
• Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
• Add 4 ul of Enzyme Master Mix
• Digest 37C/30 min, heat kill 80C/20 min

• Ligation

• Add 2ul of digested plasmid backbone (25 ng)
• Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
• Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
• Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
• Add 0.5 ul T4 DNA ligase
• Add water to 10 ul
• Ligate 16C/30 min, heat kill 80C/20 min
• Transform with 1-2 ul of product

Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

• PCR mix

• 100 ul PCR Supermix High Fidelity
• 0.7 ul of SB-prep-3P-1
• 0.7 ul of SB-prep-2Ea
• 0.5 ul template DNA at 10 ng/ul

Notes: Do not use a sample of linearized plasmid backbones (PCRed) as a template, The Registry uses BBa_J04450 as a template

• PCR program

• 94C/2min
• 94C/30s
• 55C/30s
• 68C/3min
• Repeat cycle (steps 2 to 4, 35 more times)
• 68C/10min
• Digest with DpnI enzyme: 2ul in 100ul reaction, incubate 37C/hour; heat kill 80C/20min

• PCR cleanup

• QIAquick PCR Purification
• Add 500 ul Qiagen buffer PB
• Spin through a column twice, discard flowthrough
• Wash 1x with 700 ul buffer PB
• Wash 2x with 760 ul buffer PE
• Discard liquid, spin dry at 17000g for 3 min
• Elute into a new tube twice with 50 ul of TE (100 ul total)

• Quality Control

• We recommend QCing constructed linearized plasmid backbones, to test success of PCR, ligation efficiency, and background.
• Run unpurified PCR product (1 ul) on a gel to verify the correct band and concentration and lack of side products.
• Test concentration of purified PCR product. Note: Expected yield should be 40ng/ul or higher. Adjust to 25ng/ul with TE.
• Run a digest and ligation test with purified PCR product to determine EcoRI and PstI cutting and ligation efficiency.

• Digest

• Digest Master Mix (10rxns)
• 15 ul NEB Buffer 2
• 1.5 ul BSA
• 90 ul dH20
• Run Digest
• 4 ul of plasmid backbone (approximately 100 ng)
• 10.5 ul of Digest Master Mix
• 0.5 ul either EcoRI-HF or PstI enzyme (not both!)
• Digest 37C/30min; 80C/20 min
• Proceed directly to ligation

• Ligation

• Ligation Master Mix (10rxns)
• 20 ul T4 DNA ligase buffer
• 5 ul T4 DNA ligase
• 25 ul water
• Ligation Test
• Add 5 ul of ligation master mix to digested product
• Ligate 16C/30min; 80C/20 min
• Run all 20 ul on a gel
• Compare intensity of the single and double length bands. More efficient ligations will show stronger double length bands than single.

• Transformation

• Transform 1 ul of the diluted final product into highly competent cells
• Control transform 10 pg of pUC19
• Plate on the appropriate antibiotic
• Observe few colonies. Any colonies represent background to the three antibiotic assembly process
• Quantify the effective amount of remaining circular DNA able to transform

1. Miniprep your two parts.
2. Digest your two parts and construction plasmid backbonedestination vector with the following enzymes

• Left part with EcoRI and SpeI
• Right part with XbaI and PstI
• Construction plasmid backbone with EcoRI and PstI. Also digest the construction plasmid backbone with DpnI if possible to eliminate any plasmid remaining from the PCR.

1. Combine 1 ul of each restriction digest reaction with 1 ul of ligase in a 25 ul reaction.
2. Transform the ligation product.
3. If the input parts are good, almost all colonies will be correct.
4. If desired analyze the transformation with single colony PCR followed by agarose gel electrophoresis.

• In rolling, large scale assembly, this step is often omitted.

1. Miniprep clones that generated a band of the appropriate size.
2. Sequence the clone.
3. Record the sequence information in the Registry.

Some other protocols

• [http://tools.invitrogen.com/content/sfs/manuals/purelink_PCR_man.pdf PureLink PCR Purification kit ·k3-100-01]

• [http://www.neb.com/nebecomm/products/productE0546.asp Biobrick TM Assembly Kit]

• [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110111a.pdf Aurum Plasmid minikit 100 preps #732-6400]

• Extraction of DNA from Filter Paper

• [http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/Sustainable-Features-Fact-Sheets.Par.84109.File.dat/CO21121_Purelink_kit.pdf Purelink Quick Gel Extraction Kit #k2100-12]

• [http://www.promega.com/resources/protocols/technical-bulletins/101/pureyield-plasmid-miniprep-system-protocol/ Pureyield plasmid miniprep system #a1223]

• [http://tools.invitrogen.com/content/sfs/manuals/PureLink_HQMiniPlasmid_QRC.pdf Plasmid Purification Protoco]