IGEM:Stanford/2009/Project Homeostasis/Experimental Protocols

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Experimental Protocols
Resources:
 * Jerome Bonnet's PDF of Protocols [[Media:Protocols,_Jerome.pdf | Download Here]]
 * Qiagen Handbooks Search for Proper Handbooks Here

Additional Documents:
 * PCR Protocol Template starting from Primers [[Media:PCR_Protocol_Temp.pdf | Download Here]]
 * High Fidelity PCR Manual [[Media:FidelityPCR_Protocol_Temp.pdf | Download Here]]

Liquid Culture Protocol
Objection: A Liquid Culture grows up the colonies of bacteria w/ desired plasmid.


 * Add 5 mL LB broth into 25mL tubes
 * Add proper amounts of antibiotics to the LB
 * Amp 1000X so add 5ul
 * Kan 200x so add 25ul
 * Quickly dab tip on a colony from the bacterial plate and drop tip into tube.
 * Incubate at 37C on shaker for 12-16 hours

Miniprep Protocol
Objection: A Miniprep extracts the desired plasmid and allows for an assay w/ the spectrophotometer to find the concentration of your plasmid.


 * See here for the handbook for the Qiagen Spin Miniprep Kit.

Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge

This protocol is designed for purification of up to 20 &mu;g of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37. Please read “Important Notes” on pages 19–21 before starting. Note: All protocol steps should be carried out at room temperature.

Procedure


 * 1) Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 &deg;C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
 * 2) Add 250 &mu;l Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. If using LyseBlue reagent, solution turns blue.
 * 3) Add 350 &mu;l Buffer N3 and invert the tube immediately but gently 4–6 times. If using LyseBlue reagent, solution turns colorless and cloudy.
 * 4) Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
 * 5) Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
 * 6) Centrifuge for 30–60 s. Discard the flow-through.
 * 7) Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through
 * 8) Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
 * 9) Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer
 * 10) Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 &mu;l 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
 * 11) Take and record concentration readings from the nanodrop spectrometer. Make sure to blank it first!

Insert and Cloning Vector Digestion Protocol
Objective: A Digestion cuts the desired parts out with restriction enzymes and creates accepting sites in the cloning vector.

Procedure:
 * Digest with appropriate enzymes @37C
 * Heat inactivate the enzymes 20 min @ 80C
 * Time required: 1-1.5 hours
 * Materials
 * NEB Buffer 2
 * NEB BSA
 * Ultrapure Water
 * PCR tubes
 * 37C and 60 C incubators/waterbath (best is a PCR machine where you can program this protocol)
 * 1) Mix 1-3 ug plasmid with 5ul NEB bufffer 2 and 0,5 ul BSA + ultrapure water to 50ul
 * 2) Add 1 ul of each enzyme
 * 3) Mix well by pipetting up and down so that you solution is homogenous and glycerol is not in the bottom of the tube
 * 4) Incubate for 15 to 60 min @ 37C
 * 5) Heat inactivate the enzymes by heating @ 80C for 20 min
 * 6) For insert, proceed to gel purification
 * 7) For the vector proceed to dephosphorylation (if no ccdb gene inside see part Bba_P1010)

Vector Dephosphorylation Protocol
Objective: A Vector Dephosphorylation prevents self-ligation of mono-digested vector, thereby increasing the yield of positive clones. Procedure
 * Time Required: 40 min. to 1 hour
 * Materials: Antarctic Phosphatase and buffer (NEB).
 * 1) Add 10X Antarctic phosphates buffer to your digested vector (5ul for 50ul), mix well.
 * 2) Add 1ul of Antarctic phosphatase, mix well.
 * 3) Incubate @ 37C for 30min to 1 Hour.
 * 4) Heat inactivate @ 65 for 5min.
 * 5) Proceed to purification

More infos: http://openwetware.org/wiki/Phosphatase_treatment_of_linearized_vector

Insert Purification by Gel Extraction Protocol
Objective: To get rid of enzymes and salts and to acquire clean products to work with. 1.Run your digested insert on a 0,8 to 1% agarose gel. 2. Cut the band from the gel and purify it. Procedure: A. Running the insert in the gel !!! Be carreful using ethidium bromide!!!
 * Time Required: 1.5-2 hours
 * Materials
 * Loading Buffer
 * 100bp DNA ladder
 * Agarose gels material
 * UV table
 * falcons 15ml
 * 50C Heating block.
 * Qiagen gel extraction kit (please read the protocol)
 * 1) Add 1/6 loading buffer to your digested insert.
 * 2) Place the gel in the tank (DNA will migrate toward the (+) red pole)
 * 3) Add 1X TBE buffer to cover the gel
 * 4) Remove the comb
 * 5) Load your sample on a 1% gel
 * 6) Add the 100bp DNA ladder in a another comb
 * 7) Close the Lid, plug on the generator.
 * 8) Push start and set the voltage around 50 to 90 V.
 * 9) Run for 30/45min: look at the blue marker
 * 10) Take the gel out of the tank place it on a paper towel for transport

B. Cutting the insert out BE CAREFUL USING THE UV. ALWAYS PROTECT YOUR EYES AND SKIN !!!! Use the screen and wear goggles.
 * 1) Put the gel on the UV table
 * 2) Turn on the table on the low intensity position
 * 3) Locate your band
 * 4) With a clean razor blade, cut the gel around the band (fast)
 * 5) Turn off the UV
 * 6) Take the band off, put it into a 15ml falcon

C.	Insert purification
 * 1) Add buffer QG (Orange) to your gel band
 * 2) Heat @ 50C, and vortex every 5min, the gel will dissolve
 * 3) Purify using a mini-elute Qiagen column
 * 4) Elute with 12ul, use 1ul to quantify using the nanodrop.

Vector/PCR Products (Column) Purification Protocol
Objective: To get rid of enzymes and salts and to acquire clean products to work with. Procedures:
 * Dilute your sample on binding buffer PB
 * Purify using Qiagen column
 * Time Required: 15 minutes
 * Materials:
 * Qiaquick PCR purification kit
 * for PCR products, use the mini-elute kit (remember to keep the column at 4C)
 * 1) Dilute your sample in 5 vol of binding buffer PB (250ul for 50ul reaction)
 * 2) Mix well
 * 3) Load on the appropriate column
 * 4) Centrifuge 1min @ 13000 RPM
 * 5) Discard flowthrough, add 750ul PE washing buffer
 * 6) Centrifuge 1min @ 13000RPM
 * 7) Discard flowthrough, centrifuge 1min @ 13000 RPM
 * 8) Place column on a clean, labeled tube
 * 9) Add 12ul (mini-elute) or 30ul (standard) of Elution buffer EB on the membrane
 * 10) Let the column stand for 1min on the bench
 * 11) Centrifuge 1min @ 13000 RPM
 * 12) Quantify DNA with the nanodrop

Ligation Protocol
Objective: To put your insert into your vector using DNA ligase enzyme. Procedure: •	Control ligation are performed with H2O instead of insert.
 * Time Required: 15 min.
 * Materials:
 * T4 DNA ligase (NEB): -20C
 * T4 DNA ligase buffer (NEB). Make Aliquots of 10ul as the ATP inside the buffer can degrade after multiple thaw/freeze cycle. -20C
 * 1) Calculate your product concentration (DNA= 660 g/mol)
 * 2) For each ligation, a 1:3 and 1:5 vector to insert molar ratio is used in each experiment (2nM final vector concentration for 6nM to 10 nM insert) in a 10ul volume. Both ratios generally give good results. Start with 1:3.
 * 3) Perform ligation 10-30 min RT on your bench.
 * 1) Heat inactivate @ 65 for 20min
 * 2) Proceed to transformation

Bacterial Transformation Protocol
Objective: To place your cloned DNA inside bacterial cells that will amplify it. Procedure
 * Time Required: 1.5-2 hours
 * Materials
 * Chemically Competent cells: -80C
 * water bath 42C
 * Ice
 * Your ligation reaction
 * Add 1 or 2ul of the ligation reaction to a tube of E.coli competent cells.
 * 1) Incubate on ice for 30/45 min hour.
 * 2) Bacteria are then heat-shocked for 45 sec @ 42°C in a water bath, then returned onto ice for 10 min.
 * 3) Add 250 ul SOC medium and incubate cells for 1 hour at 37°C (shake vigorously).
 * 4) Plate on LB-Agar plates supplemented with the appropriate antibiotic and incubate overnight at 37°C.
 * Note : you can often shorten the reaction times to 30/5/30, or even 15/2/30.

Plating Following Transformation Protocol

 * 1) Pour all ~350ul onto plate w/ proper antibiotics and then add beads
 * 2) Shake sideways in numerous directions, but not in a circle
 * 3) Remove the beads
 * 4) Parafilm the plates and incubate at 37C for 12-16 hours
 * Note, performing this by a flame is advisory

Polymerase Chain Reaction (PCR) Protocol
Ojective: To amplify your insert or verify its presence Procedure: A. Insert amplification
 * Time Required: 3 hours
 * Materials:
 * Taq platinum PCR mix
 * primers
 * Ultrapure Water
 * PCR tubes
 * PCR machine
 * For insert amplification, use the Taq Platinum HiFi mix. for colonies screening, use the standard one.
 * Insert are amplified by PCR in the following conditions:
 * For a 50 ul PCR reaction:
 * Taq platinum mix, -45ul
 * 10-50 ng of template DNA
 * 20µM primer 1--1,25 ul
 * 20µM primer 2--1,25ul


 * Run PCR according to the size of your insert for elongation time and annealing temperature according to the Tm ( actually, if you start from plasmid, you can significantly decrease the annealing Temperature without significant background; I actually run most of my PCR @ 55C). Elegation temperature for Taq is 68C
 * Amplification is verified by agarose gel electrophoresis, the full sample is run on a 1% Agarose gel or 2% for inserts smaller than 1Kb.

Colony Screening Protocol
Objective: To Find the cells containing your cloned DNA Procedure
 * Materials:
 * PCR machine
 * Taq platinum PCR mix (not HiFi)
 * Biobricks primers
 * Gel material
 * 1) Ligations generally yield about 50 times more colonies per ligation than on control plates.
 * 2) The presence of the insert is assessed by direct PCR of colonies picked from plates, and/or by digestion of a DNA miniprep prepared from the isolated colonies.
 * 3) Pick a colony, and soak the tip in a PCR reaction mix containing the appropriate primers (biobrick primers OK); then throw the tip in tubes containing LB + antibiotic and grow cells overnight.
 * 4) Run PCR samples on Agarose gel to verify the presence of the insert.
 * 5) Perform a miniprep from 1ml of bacterial culture grown in LB from an isolated colony; Digest with the restriction enzymes (facultative if PCR OK). The presence of the insert is determined by agarose gel electrophoresis (you should see the band corresponding to the linearized vector and the band corresponding to the insert). Test several colonies for each ligation (start with 2/3 if your negative ligation control is clean).
 * 6) Sequence positive clones using appropriate primers.

Assays
[[Media:PQ_protocol.doc | PQ Assay w/ GFP... still drafting]]

“‘Protocol for PQ Assay”’


 * Previous Night:
 * Grow 5mL liquid cultures of:
 * 1) Desired plasmid w/ SoxR/S and GFP tag to be diluted
 * 2) Desired plasmid w/ SoxR/S and GFP tag to remain concentrated (3 tubes)
 * 3) Treat each w/ PQ at different concentrations- 50ul, 100ul, 500ul
 * 4) Empty Plasmid- GFP w/ no Promoter = (-) control
 * 5) Plasmid w/ Constitutive Promoter plus GFP = (+) control
 * Grow on 37C shaker 12-16 hours


 * Day of Assay:
 * 1) 	Make a stock solution of LB + Antibiotic Resistance (AMP)
 * 2) 	Ratio of 1mL:1uL
 * 3) 	Fill the plate columns to be incubated w/ PQ w/ diluted cultures
 * 4) .	In each Plate column
 * 5) 	1mL Stock Solution
 * 6) 	10 uL culture (to Dilute ~100-fold)
 * 7) 	Desired PQ amounts
 * 8) Don’t forget about controls!
 * 9) 	Columns w/ controls… make several rows of each (~3)
 * 10) 	LB
 * 11) 	(-) control… no GFP
 * 12) 	(-) control… GFP no promoter
 * 13) 	(+) control… GFP w/ promoter
 * 14) 	0uL PQ w/ Diltued cells
 * 15) 	50 uL PQ w/ Diltued cells (50uM PQ)
 * 16) 	100 uL PQ w/ Diltued cells (100uM PQ)
 * 17) 	500 uL PQ w/ Diltued cells (500uM PQ)
 * 18) 	Incubate and shake about 45 min.
 * 19) 	Transfer 200 uL of each cell into plate reader and record orientations
 * 20) 	Run proper protocol on plate reader