Difference between revisions of "Team:UPO-Sevilla/Protocols"

Revision as of 16:57, 4 October 2016

Plasmid DNA extraction

Collect the saturated bacteria culture in a 1'5 mL tube and centrifuge at 13000 rpm for 1 minute. Do it twice
Add 100 μL of GTE (Tris HCl 25 mM, pH 8, glucose 50 mM, EDTA 10 mM), mix by inverting and incubate on ice for 5 minutes
Add 150 μL of potassium acetate 3 M (pH 4'8), mix by inverting and incubate on ice for at least 5 minutes
Centrifuge the tubes at 13000 rpm for 10 minutes and transfer the supernatant to a 1'5 mL tube
Add 400 μL of ice ethanol 96% and centrifuge at 13000 rpm for 10 minutes
Discard the supernatant, add 500 μL of ethanol 70% and centrifuge at 13000 rpm for 5 minutes
Let the tubes dry and resuspend the plasmid DNA in 50 μL of TER (TrisHCl 10 mM, pH 8, EDTA 1 mM, RNAse 20 mg L ^-1)

DNA electrophoresis

GEL MAKING

Prepare 40 or 100 mL of TAE (Trisacetate 40 mM, pH 7’7, EDTA 10 mM), it depends on the size of the gel
Add 0’4 g or 1 g of agarose to the TAE solution (for 1% gel)
Heat the solution until all the agarose has been dissolved and cold it to 50ºC
Pour the the solution in the electrophoresis vessel, apply the combs and let it polymerize

GEL RUNNING

Add 1 μL of loading buffer 6x (TrisHCl 5 mM, pH 8, EDTA 0’5 mM, glycerol 30%, xylene cyanol 2’5 g L ^-1, bromophenol blue 2’5 g L ^-1) to 5 μL of DNA sample
Remove the combs from the gel, put the vessel on a horizontal electrophoresis system and cover it with TAE
Pipette 6 μL of the DNA samples and ladder
Run at 120 mV for 45 minutes approximately

REVEAL THE GEL

Put the gel in ethidium bromide for 20 minutes
Look the gel on the transilluminator and take a photo of it

Ligation

Add 6 μL of insert DNA
Add 1 μL of vector DNA
Add 2 μL of ligase buffer high ATP (TrisHCl 50 mM, pH 7’6, MgCl 2 10 mM, DTT 5 mM, BSA 50 μg mL ^-1)
Add 1 μL of T4 DNA ligase
Incubate at 16ºC overnight

Generate competent cells

Plate Escherichia coli DH5α and incubate at 37ºC overnight
Pick a colony, inoculate in LB media and incubate at 37ºC and 180 rpm overnight
Dilute from 100 to 500 times the culture in SOB media(tryptone 20 g L^-1, extract of yeast 5 g L^-1, NaCl 8’5 mM, KCl 1’25 mM, MgCl₂ 10 mM) and incubate at 20ºC until a DO₆₀₀=0’6
Cold the culture on ice for 10 minutes and collect the cells by centrifuging at 4ºC and 3000 g for 10 minutes
Discard the supernatant, resuspend in 80 mL of iced TB (PIPES 10 mM, CaCl 2 15 mM, KCl 250 mM, MnCl₂ 55 mM, pH 6’7) and incubate on ice for 10 minutes
Centrifuge at 4ºC and 3000 g for 10 minutes, discard the supernatant and resuspend in 20 mL of TB
Add DMSO (dimethyl sulfoxide) until a final concentration of 7% (v/v) and incubate on ice for 10 minutes
Distribute the cells to samples of 100 μL and store at 80ºC

Heat-shock transformation

Take the competent cells, E. coli DH5α, from the storage at 80ºC and defrost them on ice
Add 1 μL of plasmid solution to the 100 μL cell tube or add 100 μL of cells to the 10 μL ligation tube
Incubate on ice for 30 minutes
Heat-shock the cells at 42ºC for 45 seconds
Add 1 mL of SOC rich media (supplemented SOB media with 0’36% of glucose) and incubate at 37ºC for 1 hour
Plate the culture on LB-agar plate overnight

Electrocompetent cells

Grow a single Pseudomonas putida colony in LB overnight at 30ºC and 180 rpm
Harvest the cells in a 1’5 mL tube by centrifugation (30 seconds at 13000 rpm) and resuspend in 1 mL of sucrose 300 mM. Do it twice
Resuspend the cells in 100 μL of sucrose 300 mM

Electroporation and transposition

ELECTROPORATION

Use 100 μL of electrocompetent cells for electroporation
Add on ice 500 ng of the plasmid pTNS2 (allow the transposition of the plasmid pUC18SfiminiTn7BBGm) and 500 ng of the derivative plasmid of the pUC18SfiminiTn7BBGm
Transfer the reaction mix to 2 mm electroporation cuvette that has got cold previously
Electroporate at 2’5 kV, 25 μΡ and 200 Ω with a pulse of 5 ms
Add 1 mL of SOC media and incubate at 30ºC for 1 hour
Plate the culture on LB-agar plate overnight at 30ºC

TEST THE TRANSPOSITION

Do a colony PCR like a simple PCR, but adding a colony instead of a template DNA and using the primers VR and VF2
Do a electrophoresis of the colony PCR at 1'5% agarose gel
Reveal the agarose gel

Triparental mating

Grow the donor strains, the acceptor strains and the strain that contains the conjugative plasmid pRK2013 in LB overnight
Mix 300 μL of each strain (donor, acceptor and conjugative) in a 1’5 mL tube and centrifuge at 13000 rpm for 1 minute
Wash the pellet with 1 mL of LB and centrifuge at 13000 rpm for 1 minute twice
Resuspend the pellet with 100 μL of LB
Spot 100 μL of the mixed cells onto a LB-agar plate and incubate for 6 hours at the adequate temperature
Take some biomass of the spot and plate onto a LB-agar plate with the specific antibiotics of the donor plasmid and incubate overnight

PCR (iPfu^®)

Reaction Mix

PCR machine program

Digestion (NEB enzymes)

Prepare the reaction mix

Incubate at 37ºC for 2 hours

Site-directed mutagenesis using overlap extension PCR

Reaction Mix to get the two DNA fragments of the sequence that we want to mutagenize (one of the primers has the mutagenic sequence)

PCR machine program

Test the length of the PCR products in an agarose gel, excise the DNA fragment of interest and purify the agarose slice containing the DNA fragment
Run 5 μL of the purified DNA fragment solution in an agarose gel to compare the concentration of DNA for each pair of fragments
Equal the concentration of each pair of DNA fragments
Reaction mix to get the full sequence with the specific mutation

Do the same PCR machine program

Dilution series-based growth curves

Plate the strains of Pseudomonas putida KT2442 and incubate at 30ºC overnight
Grow a single colony in 3 mL of LB without antibiotics at 30ºC and 180 rpm overnight
Dilute 20 times the culture in 1 mL of LB and measure the OD₆₀₀
Adjust the OD₆₀₀ to 0’1 for 6 mL of LB
Do 9 serial dilutions with a dilution factor of 6 (take 1 mL of the culture and mix with 5 mL of LB). Induce the expression in this step, if it would be necessary
Prepare a microtiter dish for each strain and condition. Load the first and the last column with 150 μL of the media and 150 μL of the dilutions in the rest of columns, serially
Incubate the microtiter dish at 25ºC and 150 rpm for 20 hours
Measure the OD₆₀₀ of the microtiter dish (planktonic growth)
Remove the planktonic bacteria and wash the plate with water (submerge the plate in a tray of water and shake out the liquid). Repeat this step three times
Dry the plate and add 200 μL of 0’1% crystal violet solution into each well and stain for 15 minutes at RT
Discard the crystal violet solution and wash the plate in a tray with water three times
Dry the plate and add 200 μL of 96% ethanol and stain for 20 minutes and 600 rpm at RT
Measure the OD₆₂₀ of the microtiter dish (biofilm growth)

Beta-galactosidase assay

Plate the strains of Pseudomonas putida KT2442 that contains the plasmid with lapZ and the promoter that we want to test, and incubate at 30ºC overnight
Grow a single colony in 3 mL of LB with the plasmid antibiotic at 30ºC and 180 rpm overnight
Dilute 300 times the culture in 3 mL of LB with the appropriate antibiotic. Induce in this step, if it would be necessary
Incubate at 30ºC for 24 hours
Prepare the reaction mix: 705 μL of buffer Z + β-mercaptoethanol, 30 μL of Chloroform, 15 μL of SDS (Sodium Dodecyl Sulfate) and 50 μL of culture diluted 20 times (1140 μL of phosphate buffer 1x and 60 μL of culture)
Measure the OD₆₀₀ of the diluted culture
Mix the reaction mix by vortex and cool down at 30ºC. Add 200 μL of ONPG (ortho-Nitrophenyl-β-galactoside), mix by vortex and incubate at 30ºC
Add 500 μL of NaCO₃ 1M, when the reaction mix becomes yellow, and write down the time
Centrifuge at 10000 rpm for 15 minutes and measure the A₄₂₀ of the reaction mix