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Team:SDU-Denmark/protocols
From 2010.igem.org
Any deviation from these protocols will be noted in the Labnotes
Protocols
Colony PCR
CP1.1
How to amplify DNA from bacterial colonies and solutions
Important remarks
All solutions should be kept at ice until run of PCR
Materials
Premix Pfu-PCR
For 1 PCR reaction:
• 5 µL Pfu-buffer + MgSO4
• 1.5 µL 10mM dNTP mix
• 1.5 µL 10pmol/µL forward primer
• 1.5 µL 10pmol/µL reverse primer
• 0.5 µL Pfu polymerase enzyme (add just before PCR run)
• 40 µL H2O
Total vol.: 5O µL
Premix TAQ-PCR (no proofreading):
For 1 PCR reaction
• 2.5 µL 10x TAQ-buffer + MgCl2
• 0.5 µL 10mM dNTP mix
• 1.25 µL 10pmol/µL forward primer
• 1.25 µL 10pmol/µL reverse primer
• 19.25 µL Water
• 0.25 µL TAQ polymerase enzyme (add just before PCR run)
Total vol.: 25 µL
The TAQ polymerase has no proofreading, and should therefore only be used for size determination of DNA fragments. When PCR-product is to be purified and used for further experiments always use Pfu polymerase!!!
Make enough premix for your number of colony +1.
Protocol
Colony PCR:
1. Select and transfer a single colony to a PCR tube with a pipette tip (afterwards use the same for plating out on plates)
2. Add all of the H2O used in the premix to the PCR tubes and place them in the microwave at full power for 2 min. with an open lid.
3. Make the premix (without water). Do not add enzyme until just before premix is added to the PCR tubes. Mix the premix by pipetting up and down (do not vortex!)
4. Add premix to each PCR tube.
5. Run PCR
PCR of DNA in solutions:
1. Transfer 1-2 µL of DNA to each PCR tube (to obtain the correct total volume adjust the volume of the H2O)
2. Make the premix(do not add enzyme until just before premix is added to the PCR tubes). Mix the premix by pipetting up and down (do not vortex!)
3. Add premix to each PCR tube.
4. Run PCR.
PCR program:
Other programs have been used as well
Pfu-PCR
| 1 |
Start |
95°C |
2 min |
| 2 |
Denaturing |
95°C | 30 sec |
| 3 |
Annealing |
54°C |
30 sec |
| 4 |
Elongation |
72°C |
1 min |
| 5 |
GOTO 2 |
rep. 29x |
|
| 6 |
End |
72°C |
5 min |
| 7 |
Hold |
4°C |
TAQ-PCR
| 1 |
Start |
94°C |
2 min |
| 2 |
Denaturing |
94°C | 1 min |
| 3 |
Annealing |
55°C |
1 min |
| 4 |
Elongation |
72°C |
2min |
| 5 |
GOTO 2 |
rep. 29x |
|
| 6 |
End |
72°C |
5 min |
| 7 |
Hold |
4°C |
CP1.2
Updated Taq protocol for determination of product size. Due to Taq's lack of proofreading, only use this protocol for determining sizes.
Protocol:
1. A single colony is transfered to each eppendorf tube with a pipette tip. (The same tip is used to plate out on a LA+antibiotic plate afterwards)
2. Add 30 ul H2O to each tube.
3. Microwave with open lid at full power for 2 minutes.
4. Prepare Pre-Mix (number of colonies+1) Distribute 19ul to each tube.
5. shortly spin down PCR tubes
6. Load and set PCR machine
7. Add TAQ polymerase at last moment. Make sure to get it under the surface of the solution.
8. Run PCR reaction.
Pre-mix:
5ul 10x TAQ buffer
2ul MgCl2 (Increase in 0.25ul incriments if the DNA you want to extract is longer than 3kb.)
2ul 10pmol/ul forward primer
2ul 10pmol/ul reverse primer
1ul 10mM dNTP mix
7ul H2O
1ul TAQ polymerase -> NB! Pre-Mix is made without TAQ polymerase!
PCR program
| 1 |
Start |
94°C |
2min |
| 2 |
Denaturing |
94°C | 1min |
| 3 |
Annealing |
55°C |
1min |
| 4 |
Elongation |
72°C |
2min |
| 5 |
GOTO 2 |
rep. 29x |
|
| 6 |
End |
72°C |
3min |
| 7 |
Hold |
4°C |
- |
Elongation time is adjusted according to the length of the template. (1 min for every 1Kb)
CP1.3
Updated Taq protocol for size determination. Due to Taq's lack of proofreading, only use this protocol for size determination.
Protocol:
1. A single colony is transfered to each eppendorf tube with a pipette tip. (The same tip is used to plate out on a LA+antibiotic plate afterwards)
2. Add 15 ul H2O to each tube.
3. Microwave with open lid at full power for 2 minutes.
4. Prepare Pre-Mix (number of colonies+1) Distribute 9.5ul to each tube.
5. shortly spin down PCR tubes
6. Load and set PCR machine
7. Add TAQ polymerase at last moment. Make sure to get it under the surface of the solution.
8. Run PCR reaction.
Pre-mix:
2.5ul 10x TAQ buffer
1ul MgCl2 (Increase in 0.25ul increments if the DNA you want to extract is longer than 3kb.)
1ul 10pmol/ul forward primer
1ul 10pmol/ul reverse primer
0.5ul 10mM dNTP mix
3.5ul H2O
0.5ul TAQ polymerase -> NB! Pre-Mix is made without TAQ polymerase!
PCR program
| 1 |
Start |
94°C |
2min |
| 2 |
Denaturing |
94°C | 1min |
| 3 |
Annealing |
55°C |
1min |
| 4 |
Elongation |
72°C |
2min |
| 5 |
GOTO 2 |
rep. 29x |
|
| 6 |
End |
72°C |
3min |
| 7 |
Hold |
4°C |
- |
Elongation time is adjusted according to the length of the template. (1 min for every 1Kb)
--Tipi 07:40, 22 July 2010 (UTC)
Making competent cells of E. coli for transformation
CC1.1
How to make competent cells for transformation
Compotent cells enough for 12 transformations
Important remarks
Use 2 ml eppendorf tubes
Cool eppendorf tubes at 4°C prior to use
Cool 50 ml 50 mM CaCl2 at 4°C prior to use
Materials
• Overnight culture of TOP10 cells in LB media
• Ice cold 50mM CaCl2
• LB media (pre-heated to 37°C)
Protocol
1. Dilute the culture to OD550 = 0,02 in 110 ml of LB. Incubate at 37°C with shaking until OD550 reaches 0.5
2. Divide the cells in 2x55 ml and transfer to Falcon tubes (the can hold only 55 ml). From now on proceed with the 2 tubes in parallel
3. move the CaCl2 to -20°C
4. Harvest cells by centrifugation at 4100 rpm (2160 G) at 4°C for 10 min.
5. Discard the supernatant (keep the cells on ice!)
6. Resuspend cells gently in 5 ml ice cold CaCl2 (50 mM) taken from -20°C and kept on ice.
7. Repeat the centrifugation step.
8. Discard the supernatant and resuspend cells in 1.2 ml of icecold CaCl2 (keep the cells on ice!)
9. Leave the cells on ice for 30 min => now the cells are ready for transformation.
Transformation
TR1.1
Important remarks
Pre-heat LB media to 37°C
Pre-dry LA plates with the appropriate antibiotics.
Pre-cool 2 mL eppendorf tubes.
Keep cells on ice at all times!!
Remember controls:
Positive control with your uncut vector
Negative control with no inserted DNA
Materials
• Freshly made compotent E. coli cells.
• LA plates with appropriate antibiotics
• LB media
Protocol
1. Transfer 5 µl DNA (plasmid or ligation mix) to precooled eppendorf tubes. (Use only 1ul if DNA is taken from distribution plates.)
2. Transfer 200 µl of competent E. coli cells to the tube and mix by pipetting up and down (keep the cells on ice at all times)
3. Leave on ice for 30 min.
4. Heat shock for 90 sec. at 42°C in a water bath, do not shake tubes.
5. Place on ice for 2 min.
6. Add 1.5 mL of preheated LB media (37°C)
7. Incubate at 37°C for 1 hour with gentle shaking.
8. Plate 2 plates with 150 µl mixture on LA plates with the appropriate antibiotics.
9. Pellet the remaining cells 5 min at 3500 rpm and discard approximately 900 µl of the supernatant.
10. Resuspend cells and plate out on LA plates with appropriate antibiotics.
11. Incubate all plates ON at 37°C
Restriction digest
RD1.1
How to digest DNA using Fermentas fast digest restriction enzymes.
Important remarks
Remember to load 2-5µL uncut product next to the marker and take a picture of this for later documentation.
Materials
Restriction mixture:
For 1 digest reaction.
• 12 µL H2O (or 13 µL if only one restriction enzyme is used)
• 1 µL enzyme A
• 1 µL enzyme B
• 2 µL Fast Digest green buffer
• 5 µL PCR product
Multiply restriction mixtures if more digested PCR product is needed
Protocols
1. Cast an agarose gel of suitable percentage for purification of the cut product
2. Mix the restriction mixture in en eppendorf tube by pipetting up and down
3. Leave for 5 min. at 37°C (no shaking!)
4. Immidiately load the restriction mixture in the gel
5. Run the gel and cut out and purify correct sized bands.
Ligation
LG1.2
How to assemble DNA biobricks
Materials
Ligation mixture:
For 1 ligation reaction
• 2 µL 10x T4 DNA ligase buffer
• 1 µL T4 DNA ligase (add last!)
• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks
• Add H2O to reach a total volume of 20µL
Protocol
1. Prepare the ligation mixture and mix by pipetting up and down
2. Leave the mixture overnight at 17°C
2a. If there is no time leave the ligation solution at 22.5°C for 30mins. Then denature the ligase at 65°C for 10min.
3. Use ligation solution for transformations
--Tipi 06:48, 20 July 2010 (UTC)
DNA extraction from gel
DE1.1
Gel extractions were done according to the
protocol of Fermentas with the exception that we introduced an additional centrifugation step after washing to remove surplus ethanol.
DE1.3
Gel extractions were done according to the protocol of GE Healthcare with the exception that we introduced an additional centrifugation step after washing to remove surplus ethanol.
Genomic DNA purification
GP1.1
How to extract and purify genomic DNA
Important remarks
All steps should be carried out at room temperature.
Be sure to mix thoroughly when adding the solutions.
Addition and removal of chloroform should be carried out in a fume hood.
Materials
• Lysis solution
• Chloroform
• Precipitation solution (80 µL is diluted in 720 µL of H2O just prior to use)
• 1.2M NaCl solution
• Ice cold ethanol (70%)
• H2O
Protocol
1. Mix 200 µL of sample (overnight culture) with 400 µL Lysis solution and incubate at 65°C for 5 min.If a frozen sample is used lysis solution should be added before thawing and incubated at 65°Cfor 10 min. with occasional inverting the tube.
2. Immediately add 600 µL of chloroform, gently emulsify by inversion (3-5 times) and centrifuge the sample at 10.000 rpm for 2 min.
3. Prepare precipitation solution.
4. Transfer the upper aqueous phase containing DNA to a new tube and add 800 µL of freshly prepared precipitation solution, mix gently by several inversions at room temperature for 1-2 min. and centrifuge at 10.000 rpm for 2 min.
5. Remove supernatant completely (do not dry) and dissolve DNA pellet in 100 µL of 1.2M NaCl solution by gentle vortexing (make sure that the pellet is completely dissolved) To avoid loosening the pellet, keep the tube in the same angle as when placed in the centrifuge!
6. Add 300 µL of cold ethanol, let the DNA precipitate (10 min. at -20°C) and spin down (10.00 rpm, 3-4 min.).Pour off the ethanol and dissolve DNA in 15 µL of sterile dH2O by gentle vortexing.
7. Measure DNA concentration on nanodrop.
8. Store DNA at -20°C
Plasmid miniprep kit
MP1.1
Plasmids are isolated from cultures by transferring 5 mL overnight culture to a 15 mL falcon tube and spinning down at 4000g for 15 min and removing supernatant.
Then the GeneJet Plasmid miniprep kit from Fermantas was used according to manufacturers recommendations.
MP1.2
Plasmids are isolated from cultures by transferring 10 mL overnight culture to a 15 mL falcon tube and spinning down at 4000g for 15 min and removing supernatant.
Then the GeneJet Plasmid miniprep kit from Fermantas was used according to manufacturers recommendations.
MP1.3
Plasmids are isolated from exponential growing cultures by:
1. Transferring 2.5mL overnight culture to 15mL preheated LB medium and growing cells for an additionally 2 hours.
2. Transfer all 17.5mL new culture to a 50mL falcon tube and spin down at 4000g for 15 min.
Proceed with the the GeneJet Plasmid miniprep kit from Fermantas was used according to manufacturers recommendations.
Preparation of Agarose gelmix for gel electrophoresis
AG1.1
Important remarks
Agarose concentration is dependent on the size of the DNA fragment that needs to be seperated (see the door of the incubator in the gel room)
Addition of EtBr is carried out in a fume hood.
Materials
• Seachem Agarose
• TAE buffer
• EtBr
Protocol
1. For a 1% agarose gel mix 3 g agarose and 300 mL TAE buffer in a 500 mL flask.
2. The mixture is heated for 5 min. at max temperature in micro-wave. Remember to note name, date and –EtBr on the flask.
3. Place flask in the incubator for 20 min or at room temperature until cooled to 60°C.
4. Add 5 droplets of EtBr and mix.
5. Cast gel and leave for 20 min until the gel is set. Remaining agarose solution is placed in incubator for later use.
6. Load and run gel. Load 5 µL of DNA marker
Extraction af Carotenoids and polyene chromophores
EX1.1
1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours
2. Harvest cells by centrifugation at 4000g for 15 min
3. Resuspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant
5. Measure absorbance using UV-Vis spectrophotometer at 450 nm
EX1.2
1. Incubate E. Coli in 110 ml LB media with appropriate antibiotics at 37 degrees celcius for 20 hours
2. Harvest cells by centrifugation at 4000g for 15 min
3. Re-suspend cells in 8 mL acetone and sonicate the sample for 2x 30 sek
4. Centrifuge the samples at 16000g for 2 min and collect 2 mL of the supernatant
5. Measure absorbance using an HPLC at 450 nm for bata-carotene and 382 nm for retinal analysis, For this particular purpose, we use a Poroshell 120 EC-C18 (4,6 x 150 mm 2,7 micron)column, and the eluents used are as follows:
A-buffer: 100% methanol with 0,1% trifluoroacetic acid.
B-buffer: A mixture consisting of 60% methanol and 40% acetone with 0,1% trifluoroacetic acid.
Due to the chemical properties of beta-carotene and retinal, respectively, retinal will come through the column before beta-carotene when a gradient is run from 100% A-buffer to 100% B-buffer.
Afterwards, the solutions of purified retinal or beta-carotene are studied using UV-vis photospectrometry and the values and spectra are compared to those of the same compounds of known concentrations. Again, this gives both qualitative and quantitative indications of whether the compound in question is present and, if it is, in what concentration. Usage of the HPLC and instruction on how to use it was kindly provided by FLINT
Photosensor characterisation
PS1.1
Materials
• Diluted LB media
• Difco Agar
• 1µM retinal (final concentration)
Swimming motility plates
1. LB media is mixed with 0.3% difco agar and autoclavated
2. The appropriate antibiotic and 1µM retinal (final concentration) is added to the autoclaved media (NB: for the media used for the control plates, no antibiotic or retinal is added)
3. Plates are cast and incubated overnight at room temperature
4. 15 minutes prior to the experiment the plates are dried at 37°C.
Sample preparation
1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony. The culture is grown overnight at 37°C and 180rpm.
2. The overnight culture is diluted in 5mL LB media containing the appropriate antibiotic to reach an OD550 of 0.02 and are incubated at 37°C and 180rpm until it reaches an OD550 of 0.5.
3. 1µM retinal (final cnocentration) is added to the culture (NB: no retinal is added to the cultures containing the control cells)
4. The tubes containing the cultures are wrapped in tin foil (creates darkness) and are subsequently grown for 2 hours at 37°C and 180rpm.
Motility assay
1. 2x2.5µL of culture is placed on each plate, and the plates are placed in a specially engineered lightbox, so that ½ of each plate is illuminated with blue light and the other ½ is kept in dark.
2. The plates are incubated at 37°C for 24 hours.
3. Pictures are taken
Microscopy
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.
3. Samples are examined under the microscope.
PS1.2
A protocol for optimizing the motility of E.coli MG1655 to use for microscopy
This protocol is designed based on preceeding pilot studies
Materials:
• LB media
• Motility buffer (20mM potassium phosphate and 0.1mM EDTA dissolved in ddH2O [1]
• 1mM retinal
Protocol:
1. A colony is inoculated in 5mL LB media with appropriate antibiotic.
2. The culture is incubated for 12h. at 22° and 160rpm
3. The cultures containing the unmodified bacteria (controls) is then diluted 1:10 in motility buffer, at which point these are ready for microscopy
4. The culture containing the modified bacteria is added 1uM retinal (final concentration), and is incubated for an additional 2h in darkness at 22°C and 160rpm.
5. The culture is then diluted 1:10 in motility buffer and are ready for microscopy.
Microscopy
1. 5µL of bacterial culture is placed in the center of a microscopy slide dimensions 7.5cm x 1.5cm and a cover slide is used to cover the culture.
2. To eliminate any flow in the system, which can be mistaken for bacterial motility, the cover slide is sealed with ordinary mail polish.
3. Samples are examined under the microscope.
Growth rate assay
GA1.1
Materials
• LB media
• Spectrophotometer
Protocol
1. 5mL LB media containing the appropriate antibiotic is inoculated with a colony .
2. The culture is incubated over night at 37°C and 180rpm.
3. The optical density at 550nm (OD550) of the overnight culture is measured and the culture is diluted in 15mL fresh LB media containing the appropriate antibiotic to reach an OD550 of 0.02. The culture is then incubated at 37°C and 180rpm.
4. OD550 of the colony is measured every hour for the first 12 hours, and after 24 hours.
Flagella staining
The following protocols are based on knowledge recived from [2]
FS1.1
Day 1: The bacteria were grown in 5 ml-LB media overnight. The solutions used for staining were prepared.
Solution I:
The following components were added in the listed order:
• 5 g of tannic acid dissolved in 9.65 ml distilled water.
• 150 µl 9% FeCl3
• 100 µl 1% NaOH
• 200 µl formalin
Solution II:
• 2 g silver nitrate was dissolved in 10 ml distilled water
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.
Day 2: The bacteria were boosted in 5 ml LB-media to ensure that they were in the exponential growth phase when used for staining. They were diluted to approximately an OD550 of 1.
Preliminary bacteria work:
• The bacteria were centrifuged 15 min at 4000rpm
• The pellet was resuspended in LB-media to an OD550 of 3.
Staining protocol:
• A clean glass slide was used and Poly-L-Lysin was added onto a small area.
• 20 µl of the bacteria solution was plated on the slide and allowed to air dry.
• The slide was flooded with solution I and allowed to stand for 30 min before it was washed with distilled water.
• Solution II was added and was incubated at room temperature for 10 min and was washed with distilled water.
• The slide was flooded with a carbol-fuchsin solution and air-dried before washed with distilled water.
PBS were added to the area containing the bacteria and they were covered with a cover slide. The slides are now ready for examination under the microscope.
FS1.2
Day 1: Bacteria were platede on agar plates and incubated at 37 degrees celcius overnight. The staining solutions were prepared.
Solution I:
The following components were added in the listed order:
• 5 g of tannic acid was dissolved in 9.65 ml distilled water.
• 150 µl 9% FeCl3
• 100 µl 1% NaOH
• 200 µl formalin
Solution II:
• 2 g silver nitrate was dissolved in 10 ml distilled water.
• 10% aqueous ammonia solution was added until the silver nitrate was dissolved. Approximately 2 ml.
Day 2: A bacterial colony was dissolved in LB-media.
Staining protocol:
• A clean glass slide was used and Poly-L-Lysin was added onto a small area.
• 20 µl of the bacteria solution was plated on the slide and allowed to air dry.
• The slide was flooded with solution I and allowed to stand for 30 min before it was washed with distilled water.
• Solution II was added and was incubated at room temperature for 10 min and was washed with distilled water.
• The slide was flooded with a carbol-fuchsin solution and air-dried before washed with distilled water.
PBS were added to the area containing the bacteria and they were covered with a cover slide. The slides are now ready for examination under the microscope.
Stability assay
SA 1.1
Stability assay
Materials
• LB media
• LA plates
• LA plates with appropriate antibiotic
• 0.9% NaCl
Protocol
1. 5mL LB media with appropriate antibiotic is inoculated with a bacterial colony.
2. The culture is incubated over night at 30°C and 180rpm
3. 100µL of the culture is serially diluted in 900µL 0.9% NaCl until a dilution of 107 is reached.
4. 100µL of the 105, 106 and 107 dilutions are spread onto LA plates, and LA plates with the appropriate antibiotic, respectively.
5. Plates are incubated at 37°C for 16-24 hours. The following day the colonies formed on the plates are counted and cfu are determined.
6. 500µL of the 105 dilution is added to 4.5mL of fresh LB media without any antibiotics.
7. The new culture is incubated over night at 30°C and 180rpm
8. The experiment is carried out for 5 days.
(NB: antibiotic is only added to the first culture. The remaining days the bacteria are grown in cultures without any antibiotics)
Scanning Electron Microscope
SEM 1.1
Day 1: The bacteria was cultivated overnight in 5 ml LB-media at 37 degrees celcius.
Day 2: The overnight culture was centrifuged 15 min at 4000prm. Afterwards the pellet was resuspended in distilled water. We aimed to get approximately 106 bacteria in 10 µl solution which was plated on double adhesive tape at the top of the grid. The solution was allowed to air dry and the remaining fluid disappeared as samples were exposed to the vacuum in the electron microscope.
The sample was examined with different electron intensity and magnification. We found that the best picture was taken with a electron intensity of 10 kV and a magnitude on 10kx.
Charactarization of K389016 (VirA/G reporter device mRFP)
CK1.1
Materials:
• 20mM acetosyringone (39.3 acetosyringone dissolved in 1mL DMSO and 9mL ddH2O)
• LB media
• 35µg/mL chloramphenicol
Protocol:
1. A colony is inoculated in 5mL LB media with 35µg/mL chloramphenicol and incubated over night at 37°C and 180rpm.
2. Parallel cultivations of 25mL LB media, 35µg/mL chloramphenicol and acetosyringone concentrations of 0uM (control), 100uM, 200uM and 400uM respectively.
3. The cultures were incubated in a waterbath of 37°C and 180rpm
4. The optical density at 550nm (OD550) was measured every two hours and samples were freezed at -80°C and used for fluorescence measurements (excitation at 584nm, emmitation at 607nm)
References
[1]Khan S, Amoyaw K, Spudich JL, Reid GP, Trentham DR,Bacterial chemoreceptor signaling probed by flash photorelease of a caged serine, Biophys J. 1992 Apr;62(1)
[2]Harshey RM, Matsuyama T,Dimorphic transition in Escherichia coli and Salmonella typhimurium: Surface-induced differentiation into hyperflagellate swarmer cells,Proc Natl Acad Sci U S A. 1994 August 30; 91(18): 8631–8635
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