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From 2011.igem.org

Team's notebook protocols

The iGEM experience is not merely a project or a conference, but it was the way that most of our students got acquainted with the world of biological research laboratory. Pipettes, solutions, gels, electrodes, dishes and other scary machinery quickly filled our lives. From day one we saw the vast importance of teaching our students to keep a proper laboratory journal. We optimized and adjusted the protocols we used during the summer, and we would like to share them to the public. Last year we have made short films of our protocols and also shared the protocols in a text form on Open Wet Ware. With this section you can see into our daily work. For detailed protocols, see below.

Protocols of Bacterial work and cloning

Making of LB medium (Lysogeny Broth, also referred as Luria-Bertani Medium)

- Scientific Background

Giuseppe Bertani published the original formula of LB, which is used for the cultivation of E. Coli. It provides the main essential nutrition, containing peptides. For transformation, we use LB agar but for small and big cultures we use the original liquid LB.

- Overview

The presparation is to dissolve LB powder in distilled (MilliQ) water, and autoclave it.

- Materials

For 1 L LB Broth

 1 L dH20 ;  20g LB powder ;  a 1 l bottle ;  measuring cylinder ; Laboratory scale

- Procedure

1) Take a jar of LB powder

2) Measure 20 g of LB powder in a scale ( it depends on the amount of LB you need, take 10 g if you’d like to make 500ml of a LB medium )

3) Put the powder into a 1 liter flask or bottle, (again, depends on how much you need)

4) Add the distilled water into the bottle till it reaches 1 liter

5) Shake the bottle carefully

6) Close it and stick a tape from one side of the wall vertically over the cap to the other side of the wall (which means not sterile) which will be black striped after autoclave sterilization

7) Sterilize the bottle (autoclave)

- References

Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.,1.25-1.28. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, USA.

Bacterial transformation by heat shock

- Scientific Background

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation (our lab tends to use a protocol based on treatment with calcium-chloride-pipes-glicerol solution). We tried several protocols last year, but we found that this is the best yielding among them. This protocol may be particularly useful if you are finding that your transformations are not working or yiedling few colonies.

- Overview

Put the DNA to be introducend and your competent cells together, change incubation temperature dramatically some times then spread the cells on LB agar plates and incubate at 37 degrees overnight.

- Materials

• Competent cells (we always use DH5α strain - [1])

• DNA to be introduced

• Crushed ice, 1,5 ml Eppendorf tubes

• 42°C water bath

• 37°C incubator

• Petri dishes with LB agar and appropriate antibiotic

- Procedure

1. Start thawing the competent cells on crushed ice (we find this cells in the -70°C fridge)

2. Put 50μl competent cells and 100-250ng DNA into a 1,5 ml tube kept on ice

3. Incubate the cells for 20 minutes on ice

4. Heat shock at 42°C for 90 seconds in water bath (do not shake!)

5. Incubate for 5 minutes on ice again

6. Spread the transformed bacteria on the Petri dishes with LB agar and the appropriate antibiotic(s) with the part name, plasmid backbone and antibiotic resistance

7. Incubate the plate at 37°C for 14 hours

- References

Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.,1.25-1.28. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, USA.

MiniPrep

- Scientific Background

This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Lysogeny Broth, Luria-Bertani) medium.

- Overview

This year we used GeneJet Plasmid Miniprep Kit (Thermo Scientific), and followed the provided protocol with some minor changes.

- Materials

• Kit containts
• table-top centrifuge at >12000 x g(10 000-14 000 rpm)
• centrifuge for 50 ml tubes with relative high performance

- Notes

All purification steps should be carried out at room temperature. All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g(10 000-14 000 rpm, depending on the rotor type). Use 1-5 ml of E. coli culture in LB media for purification of high-copy plasmids. For low-copy plasmids use up to 10 ml of culture.

- Procedure

0. [small culture] Next day after transformation of the bacteria pick up a colony with a pipette tip from the agar plate, wash into 1-5 ml LB medium (with the proper antibiotic) and put the tube into a shaker to 37°C for 12 hours. After incubation, centrifuge the tube in a adequate centrifuge at 10000xg for 10 minutes. Pour off the used medium.

1. Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain. Note. Ensure RNase A has been added to the Resuspension Solution.

2. Add 250 μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.

3. Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.

4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA. 5 Transfer the supernatant to the supplied GeneJET™ spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Step Procedure

6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Note. Do not add bleach to the flow-through, see p.7 for Safety Information.

7. Add 500 μl of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET™ spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

8. Repeat the wash procedure (step 7) using 500 μl of the Wash Solution.

9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

10. Transfer the GeneJET™ spin column into a fresh 1.5 ml microcentrifuge tube (not included). Add 50 μl of the Elution Buffer to the center of GeneJET™ spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.

11. Discard the column and store the purified plasmid DNA at -20°C.

References

Birnboim, H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1522.

MidiPrep

- Scientific Background

This protocol is designed for the preparation of High Copy Number plasmids. We used Genopure Plasmid Midi Kit (Roche).

- Materials and notes

• Sample: 50 ml E. coli culture, transformed with a high copy number plasmid. Harvest cultures at a density between 2.0 and 6.0 A600 units per ml bacterial culture.

• Media: The isolation method is optimized for cultures grown in LB media; other rich media may require increased volumes of Suspension-, Lysis- and Neutralization Buffer, and an additional wash step

• Plasmid size: The isolation procedure is suitable for all plasmid sizes; lysates of larger constructs (up to 100 kb) should be cleared by filtration to avoid shearing

• Suspension Buffer/RNase A: To dissolve the lyophilized enzyme in Suspension Buffer, pipet 1 ml of Suspension Buffer (bottle 1, black cap) into the glass vial containing the lyophilized RNase (bottle 2, black cap). Reinsert the rubber stopper and invert the vial until all lyophilizate (including any that might stick to the rubber stopper) is dissolved. Transfer the dissolved enzyme back to the Suspension Buffer bottle (bottle 1). This is enough working solution for 60 Midi preps (isolation of up to 100 μg plasmid DNA/preparation)

• If preparing aliquots of the working solution, remember that the final concentration of RNase A in the working solution must be 100 g/ml. Reconstituted buffer is stable for 6 months is stored properly (+2 to +8°C)

• Neutralization Buffer: Before starting, cool it down to 4°C

• Elution Buffer: Before starting, warm up the buffer to 50°C

• Ethanol: Use 70% ethanol. Before starting, cool it down to 4°C

- Procedure

1. Centrifuge bacterial cells from 30 ml culture grown in LB medium by centrifuging for 10 min at 5000 rpm,4°C. Discard the supernatant. Carefully resuspend the pellet in 4 ml Suspension Buffer + RNase and mix well.

2. Add 4 ml Lysis Buffer to the suspension and mix gently by inverting the tube 6 times. Incubate 2-3 min at room temperature. To avoid shearing genomic DNA, do not vortex the suspension in Lysis Buffer. To prevent release of chromosomal DNA from the cell debris, do not incubate for more than 5 minutes.

3. Add 4 ml chilled Neutralization Buffer to the suspension. Immediately mix the suspension gently by inverting the tube 6 times until a homogenous suspension is formed. Incubate the tube 5 min on ice The solution becomes cloudy and a flocculent precipitate will form.

4. Clear the lysate by filtration. Put a folded filter into a funnel that has been inserted into a 50 ml plastic tube. Moisten the filter with a few drops of Equilibrium Buffer. Load the lysate onto the wet, folded filter and collect the flowthrough. The SDS precipitates with cellular debris when Neutralisation Buffer is added; this white precipitate should not be loaded onto the column. If the solution obtained after step 14 is not clear, remove the remaining precipitate by passing the solution over a folded filter.

5. Mount the sealing ring to the column to fix the column in the collection tube. Insert one column into one collection tube. Equilibrate the column with 2,5 ml Equilibration Buffer. Allow the column to empty by gravity flow. Discard the flowthrough.

6. Load the cleared lysate from step 4 onto the equilibrate column. Allow the column to empty by gravity flow. Discard the flowthrough.

7. Wash the column with 5 ml Wash Buffer. Allow the column to empty by gravity flow. Discard the flowthrough.

8. Repeat step 7 Discard flowthrough and collection tube.

9. Re-insert the column into a new collection tube Elute the plasmid with 5 ml prewarmed Elution Buffer (50°C) Allow the column to empty by gravity flow The collection flowthrough contains the plasmid Elute the plasmid again with the flowthrough Allow the column to empty by gravity flow Plasmid concentration is higher, than after only one elution.

10. Precipitate the eluted plasmid DNA with 3,6 ml isopropanol Total volume ~6,8 ml Divide the eluted plasmid into five 1,5 ml eppendorf tubes and one tube which contains 73 μl. Centrifuge immediately 30 min at 15000×g (rcf), +4°C. Carefully discard the supernatant.

11. Divide the 6 tubes into two groups. Wash the plasmid DNA with 1,5 ml chilled (+4°C) 70% ethanol in the first tube and afterwards wash the other two with the same ethanol. Wash the other group (3 tubes) the same way.Centrifuge 10 min at 15000×g (rcf), +4°C. Carefully remove ethanol from the tube with pipet tip.Air-dry the plasmid DNA pellet for 10 min.

12. Carefully redissolve the plasmid DNA pellet in 100-200 μl TE buffer.

13. Measure the concentration of DNA with NanoDrop.

Restriction digestion

- Scientific Background

BioBrick standard biological parts are flanked by well characterized upstream and downstream sequences which are technically not considered part of the BioBrick part (aka prefix and suffix). These up/down stream segments contain restriction sites for specific restriction enzymes, which allows for the simple creation of larger BioBrick parts by chaining together smaller ones in any desired order.

In the process of chaining biobrick parts together, the restriction sites between the two parts are removed, allowing the use of those restriction enzymes without breaking the new, larger BioBrick apart. To facilitate this assembly process, the BioBrick part itself should not contain any of these restriction sites.

One such type of assemblies is the “three antibiotic” assembly standard. This assembly begins with a restriction step.

- Overview

The following protocol contains detailed instructions on the restriction digestion step of the “three antibiotic” standard assembly. It starts with a medium amount of the two parts to be assembled and a medium quantity of the backbone that the parts will be assembled into. The result is a small amount of the insert part ready to be ligated into a linearized backbone.

Note: This protocol uses NEB High Fidelity restriction enzymes and buffers.

Note: One unit of restriction enzyme cuts 1ug of DNA in 1 hour between optimal conditions.

- Materials

• DNA sample
• appropriate restriction enzyme(s)/NEB
• Nuclease Free Water
• Buffers/NEB
• occasionally BSA
• PCR tubes
• crushed ice
• waterbath or thermocycler

- Procedure

1. Prepare the following mixes in 3 different PCR strips, work on ice.

Note: the restriction of Part A, Part B and the backbone at the same reaction is not mandatory and may be split over several reactions

basic components:

                 * DNA 3-5 ug
                 * Restriction enzyme(s): 0,5 - (0,5)ul
                 * NEBuffer (1-4): 1 ul
                 * (BSA: 0,1 ul, if one of the enzymes recquires it)
                 * Nuclease free water to 10 or 20 ul.

2. Resuspend with a pipette all the components to assure proper mixing

3. Incubate at 37°C for 30 minutes

4. Run products on gel electrophoresis. (Nice bands on gel are made by 100-300ng of DNA).

5. Use PCR Purification Kit to get rid of salts and unnecessary reagents (this step is needed before every ligation)

- Notes

The volume of restriction enzymes used must not exceed 10% of the reaction volume (enzymes are dissolved in glycerol which damages the reaction).

The optimal total volume is 10 ul.

Before starting the reaction calculation it is vital to examine the enzymes information page in order to obtain:

Optimal working buffer -May cause a change in the procedure stated above

Optimal working temprature -May cause a change in the procedure stated above

- References

Sean C. Sleight, Bryan A. Bartley, Jane A. Lieviant, and Herbert M. Sauro "In-Fusion BioBrick assembly and re-engineering" Nucleic Acids Res. 2010 May; 38(8): 2624–2636.

Gel electrophoresis

- Scientific Background

Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

- Overview

After the preparation of the gel, we put DNA samples into the preformed wells with Loading Dye, than run by electric field.

-Materials

• agarose powder

• sterilized bottle

• 1x TAE solution

• microwave oven

• GelRed nucleic acid stain (non-teratogen, non-carcinogen, non-mutagen)

• laboratory scale

• 6y loading Dye (dextran/glycerol and bromothymol blue stain)

• 1 kb DNA ladder

• Gel electrophoresis apparatus

- Procedure

1. measure 1 g agarose (for 1% gel agarose).

2. put it into a sterilized bottle.

3. masure 100 ml 1x TAE.

4. put the bottle into the microwave (must not close the bottle totally), heat it still it will be fully clear.

5. Take the bottle out from the microwave with the plastic gripping.

6. Cold down the bottle in water bath until you can touch it.

7. Add 100 µl GelRed into the bottle (because 1000x must attenuate).

8. Shake gently the bottle still the red color disappears.

9. Put the liquid from the bottle into the gel tray (if there are any bubbles in it you can punch them with a tip)

10. After it became solid turn the gel rack to 90°.

11. Put 1x TAE into the gel tray still the level of line on the wall

12. Take out the comb

13. Put a ladder (3-6 µl) into the first hole

14. Into a new tube mix the loading dye(1 µl) and your sample(5 µl)

- References

Theriogenology. 2010 Sep 10 ,Huang HW, Su YF, Yao CT, Hung YC, Chen CC, Cheng CC, Li SS, Chang HW. "High-throughput gender identification of three Columbidae species using melting curve analysis"

PCR cleanup of the restricted DNA

- Scientific background

Some kits are available commercially to purify DNA or RNA from reactions like PCR, restriction digestion, dye labelling etc. Some are based on gel pufification, to clean up DNA cut from agarose gel or on the well establisher spin column technique for liquid samples. Our team used this kit for the purification of mixes after restriction digestion to get rid of salts and enzymes of the solution (the rest went on a gel).

- Overview

The method used by out team were based on the well established spin column approach including binding, washing and elution steps for purification.

- Materials

• kit contents
• restriction mix
• table-top centrifuge

- Procedure

For detailed steps see: [[2]]

Protocols from tissue culture room

Preparation of DMEM medium and PEI solution

- Scientific Background

I. The Medium

Eukaryotic cells, derived from multicellular animal eukaryotes, can be maintained in culturing media. Aside from temperature and gas mixture, the most commonly varied factor in eucaryotic culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The type of the medium used depends on the type of the cell line.

II. PEI solution

PEI (10 mM) is a cationic polymer used for PEI-mediated transfection.

- Overview

I. The Medium

For culturing cells and for ligand treatment we use DMEM (Dulbecco's Modified Eagle's Medium) completed with 10% FBS, 1% Penicillin-streptomycin and 2% L-Glutamine.

For transfections we use DMEM medium without FBS, but other constitutives are included.

The preparation procedure takes 40-50 minutes including the FBS melting time (stored in 50 ml aliquots at -20°C).

II. Preparing PEI solution

The reconstitution consists of 3 steps including dissolving PEI, adjusting pH and sterilization by filter sterilizer.

- Materials

I. For the Medium:

500 ml Basic DMEM ( Dulbecco’s modified Eagle Medium ) ordered from Sigma (D5671)

50 ml FBS (Foetal Bovine Serum)

10 ml 200 mM L-Glutamine ordered from Sigma(G7513)

5 ml 100x Penicillin-Streptomycin ordered from Sigma(P4333)

Serological pipettes, Pipettor

Sterile laminar flow box, kimwipes, 70% ethanol sprinkle bottle

II. Preparing PEI solution:

Pipette, pipet tip, glass, analitical scale

MQ water (double distilled, sterilized)

MW=25.000 Pei (ALDRICH 408727)

15, 50 ml tubes

pH measuring electrodes

HCl-solution

Sterile filter – with 0,2 um diameter pores

- Procedure

I. Preparing Medium:

1. Prepare the laminar flow box: turn on the ventillation, wait for 15 minutes, clean the bottom and the glass of the hood with 70% alcohol, and wipe it with kimwipes.

2. Put the Basic DMEM solution, the FBS, Penicillin Streptomycin and the L-Glutamine solutions into the 37°C waterbath and wait for 30 minutes (The FBS is melting slowly)

3. Take out the melted solutions from the waterbath, wipe them, squeeze them down with 70% ethanol and load them into the hood as well as the serological pipettes and the Pipettor (squeezed down)

4. Put these amounts into the basic DMEM by using a pipettor and serological pipettes:

A. For 10% FBS medium:

Basic DMEM: 500 mL

FBS: 50 mL

L-Glutamine: 10 mL

Penicillin-Streptomycin: 5 mL

B. For Serum free medium:

Basic DMEM: 500 mL

FBS: -

L-Glutamine: 10 mL

Penicillin-Streptomycin: 5 mL

5. Invert the bottle, mark it with your name, actual date and with the constituents.

6. Put the bottle to 4°C, clean up after yourself.

II. Preparing PEI solution:

1. Dissolve 4,5 mg pure PEI in 8 ml MQ water, mix well (maybe it takes one day for proper dissolvation)

2. Neutralize the solution with HCl. The final pH should be between pH 6,5-7,5

3. Adjust the volume to 10 ml

4. Filter sterilize through 0,2 um pores

5. Store the solution at 4°C.

- References

Yves Durocher, Sylvie Perret, and Amine Kamen,"High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells". Nucleic Acids Res. 2002 January 15; 30(2)

Cell subculturing (also referred as passaging")

- Scientific Background

Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).

After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.

- Notes

While working in the Cell culture lab, always follow the rules of the laboratory. You should wear a lab coat, use gloves and keep the sterile box as clean as possible.

It is advisable not to take your mobile phone into the cell culture lab.

Never bring bacterial samples into the cell culture lab!

Carefully separate dangerous waste from communal waste.

- Materials

For COS1-cells, in 10cm Petri dishes:

I. Cell passaging

• Ethanol squirt bottle

• paper towels

• 5 mL and 10 mL sterile pipets

• confluent cells in Petri dishes

• Medium (DMEM) with 10% (50ml) serum and antibiotics

• Trypsin-EDTA

• 1% PBS (Phosphate Buffered Saline)

• Pasteur Pipettes+ vacuum for aspirating the used medium

• 15 mL centrifuge tube

• Petri dishes

• Automatic pipettors

• gloves

• 37°C water-bath

• 37° thermostat

• laminar box

• tube holders

- Procedure

I. Cell Passaging for adherent culture (also called Splitting, subculturing):

1. Warm media, trypsin-EDTA and PBS in 37°C waterbath

2. Check cells in 10 cm Petri dish under Phase-contrast microscope to confirm that the cells are 90%-100% confluent

3. Clean hood with 70% alcohol

4. Sterilize all materials, bottles, etc. which are loaded into the hood. Spray hands with ethanol. Sterile pipets may be placed in the hood directly

5. Spray hands with ethanol. Remove Petri dishes from the incubator and quickly place them in the hood. (Do not spray flasks with ethanol)

6. Attach a Pasteur pipette to vacuum. Turn on vacuum system by opening vacuum valve in hood

7. Using the empty liquid media covering cells. Be careful to not touch the pipet to anything outside of the Petri dis

8. Add 2-3 mL of PBS to Petri dish. Lightly swirl PBS on base of Petri dish. Aspirate PBS from dishes

9. Add 2 mL trypsin-EDTA to Petri dish. Lightly swish trypsin

10. Place flask in incubator until detached (2-3 mins, epends on the cell-type)

11. Remove cells from incubator. Tap side of Petri dish on hard surface or your hand. Repeat several times. Visually check to ensure lumps of cells are dispersed

12. Check cells under microscope to confirm that cells are detached from the surface

13. Add 5 mL of media to dilute trypsin. Media contains antitrypsin. (Note: The liquid suspension now contains the cells.) Carefully re-suspend cells by using pipettor and pipettes

14. Aliquot appropriate volume of cell suspension into freshly prepared Petri dishes with media (The total volume in a Petri dish is 10 mL; 2mL Trypsin-EDTA, 5 ml DMEM for trypsin dilution, 3 further mL of DMEM)

15. Replace media and cells to mix. Place Petri dishes in incubator

16. Turn off aspiration

17. Dispose of liquid and solid biohazards wastes properly

18.Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean with kimwipe

1. Gerry Shaw, Silas Morse, Miguel Ararat, and Frank L. Graham Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells FASEB J. 2002 Jun;16(8):869-71