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 <table cellpadding=20px><td width="660px" height="100%" valign="top" ><p align="justify"><h2>Notebook</h2>
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-=='''Protocols'''==
-==First Week 22-24 of June==
-===Mutations===
-'''Ingredients'''
-Site directed mutagenesis of 3 plant CYP450 (79A1, 79A2, 79B1).
-We aim to destroy restrictionenzymes recognitionsites.
-Primers was supplied by IDT
-μl of 5× reaction buffer
-X μl (50 ng) of dsDNA template
-X μl (125 ng) of oligonucleotide primer #1
-X μl (125 ng) of oligonucleotide primer #2
-μl of dNTP mix
-ddH2O to a final volume of 50 μl
-Then add
-μl of X7 fusion DNA polymerase
-'''Poly Chain Reaction'''
-We ran a PCR to syntesise and amplify our mutated CYP's.
-Cycling Parameters for the QuikChange Site-Directed Mutagenesis Method
-Cycles 12
-Temperature 98°C Time 30 seconds
-Temperature 55°C Time 1 minute
-Temperature 72°C Time 30 seconds/kb of plasmid length
-'''Digestion'''
-We aim to remove the parentel CYP.
-We take advantage of the fact that this CYP is methylated on cytosines. Dpn is a restriction enzyme that cuts DNA which is methylated - therefore our new mutated CYPs remain untouched.
-. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction.
-. Gently and thoroughly mix each reaction mixture by pipetting the solution up and down
-several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and
-immediately incubate each reaction at 37°C (in a heater with lid or in a 37°C room) for 1 hour to digest the parental (i.e., the
-nonmutated) supercoiled dsDNA.
-'''Source'''
-Adapted from
-QuikChange™ Site-Directed
-Mutagenesis Kit
-INSTRUCTION MANUAL
-===Competent Cells===
-'''E. coli Calcium Chloride competent cell protocol'''
-. Inoculate a single colony into 5mL Lb in 15mL falcon tube. Grow
-O/N at 37°C.
-. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.
-. Shake at 37°C for 1.5-3hrs until OD600 = 0.4-0.8
-Then….
-. Put the cells on ice for 10 mins (keep cold form now on).
-. Collect the cells by centrifugation in the big centrifugue for 10 mins
-at 6krpm
-. Decant supernatant and gently resuspend on 10 mL cold 0.1M
-CaCl (cells are susceptible to mechanical disruption, so treat them
-nicely).
-. Incubate on ice x 20 mins
-. Centrifuge as in 2
-. Discard supernatant and gently resuspend on 5mL cold
-.1MCaCl/15%Glycerol (from a 85% stock)
-. Dispense in microtubes (300μL/tube). Freeze in -80°C.
-'''Source:'''
-Adapted from
-http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf
-===LBamp Plates===
-. 500 ml LB agar and 500 μL amphicilin/Or other antibiotic (If IPTG is needed ad it to a final concentration of 1mM, here 500 μL, which it is in the LBcam plates)
-. Pour on plates
-. Leave with the lid half on for 30 minutes at room temperature
-. Put in refrigerator until needed.
-===Transformations===
-'''Transformation of Ca++ competent cells'''
-. Put ~50μL of competent cells to prechilled microtubes. Wait 1 minute. Add 10μL (1μL if it is step 26)  of circular plasmid (c. 50 ng) or all of a ligation reaction of plasmid DNA.
-. Incubate for 15 mins on ice.
-. Heat shock for 30 seconds at 42°C. Put back on ice.
-. Add 70 μL LB
- If the resistence is cam, the sample has to incubate in an hour at 37°C while shaking.
-. Plate the whole lot in LBamp/LBcam-IPTG plates
-. Leave the plates at 37°C O/N
- If the transformation on LBcam-IPTG has succeded, the colonies are supposed to stay white.
- If they are red, the cells have not recieved the insert
-==Second Week==
-===Mini prep===
-'''Ingredients'''
-QIAprep® Spin Miniprep Kit
-* Buffer P1 (with LyseBlue)
-* Buffer P2 - Lysis Buffer
-* Buffer N3 - Neutralisation buffer
-* Buffer PB - Binding buffer
-* Buffer PE - Wash Buffer
-* Buffer EB - Elution Buffer
-* 5 ml bacterial overnight culture transformed with our BioBrick
-'''Miniprep'''
-. Pellet 5 ml bacterial overnight culture by centrifugation at 4500 rpm for 10 min at room temperature (20°C).
-. Resuspend pelleted bacterial cells in 500 μl Buffer P1 and transfer and divide in two
-microcentrifuge tubes.
-. Add 250 μl Buffer P2 to each tube and mix thoroughly by inverting the tube
-–6 times until the solution becomes blue. Do not allow the lysis reaction to
-proceed for more than 5 min.
-. Add to each 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube
-–6 times. With LyseBlue reagent, the solution will turn colorless.
-. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top
-microcentrifuge.
-. Apply the supernatant from step 5 to the QIAprep spin column by decanting or
-pipetting. Centrifuge for 60 s and discard the flow-through.
-. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
-Centrifuge for 60 s and discard the flow-through
-. Wash the QIAprep spin column by adding 0.75 ml Buffer PE.
-Centrifuge for 60 s and discard the flow-through,
-Transfer the QIAprep spin column to the collection tube.
-. Centrifuge for 1 min to remove residual wash buffer.
-. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA,
-add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the
-QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
-'''Adapted from'''
-Quick-StartProtocol QIAprep® Spin Miniprep Kit October 2010
-[http://www.qiagen.com/literature/render.aspx?id=201081]
-===Restrictionsite analysis===
-To confirm that our mutations worked we analyses the purified plasmids by cutting them with the restrictionenzyme whose recognitionsite we aim to remove.
-Ingrediens
-μl BSA (10x) diluted
-ca. 500ng Plasmid
-μl Restrictionenzyme
-μl NEBuffer
-Water up to 10μl
-Cut at 37 degress for 1 hour.
-Add 2,5μl loading buffer to the digestion
-Run it on a gel
-Visualize it and hope to see just one band. If two bands are present the mutation has not worked. (The original plasmid conatined a restrictionsite in the vector as well as in the CYP)
-==Third Week==
-===Prefix and suffix PCR Reaction===
-We add prefix and suffix to our newly made BioBricks with PCR
-'''Ingredients'''
-Water to a final volume of 50μl
-μl 5x Pfusion HF buffer
-μl 10mM dNTP
-,5μM Primer A
-,5μM Primer A
-,5 μl template (ca. 150 ng Template)
-,50 μl Pfusion X7 Polymerase
-'''PCR'''
-Initial denaturation 98°C
-Cycles 25
-Denaturation Temperature 98°C Time 10 seconds
-Anneal Temperature 55°C Time 30 seconds (The annealing temperature is determined on the basis of the sequence of the primer. Use [http://www.finnzymes.fi/tm_determination.html the calculator])
-Extension Temperature 72°C Time 15 seconds/kb of plasmid length
-Final Extension 72°C Time 10 Minuts
-'''Adapted from'''
-Finnzymes Phusion® High-Fidelity DNA Polymerase instruction guide
-===PCR purification===
-When you have added the prefix and suffix you need to purify you BioBrick. You do this by using PCR purification.
-'''Ingredients'''
-* PE buffer
-* PB buffer
-* EB buffer
-* PCR DNA
-'''Procedure'''
-. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
-. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is
-yellow.
-. Place a QIAquick spin column in a provided 2 ml collection tube.
-. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
-. Discard flow-through. Place the QIAquick column back into the same tube.
-. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
-. Discard flow-through and place the QIAquick column back in the same tube.
-Centrifuge the column for an additional 1 min.
-. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
-. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) to
-the center of the QIAquick membrane wait 1 min and centrifuge the column for 1 min.
-===Double Digest===
-Of BioBrick with prefix and suffux in order to ligate it with a plasmid.
-We have used two different approaches of the double digest. The differences are specified below as iGEM or Kenneth.
-'''Ingredients'''
-iGEM: 500 ng DNA / Kenneth: 700ng DNA
-Water until 42,5 μl (43 ul if BSA is excluded)
-μl NEB buffer (Decide which buffer: [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp])
-,5 μl BSA (Not necessary if the enzymes are HF)
-μl Restrictionsenzyme A (Decide which restriction enzymes: [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf])
-μl Restrictionsenzyme B
-Mix by flicking
-Incubate for 30 min in 37°C (BioBrick)or O/N in 37°C (Plasmid).
-iGEM: Deactivate the restriction enzymes by incubating at 80 degrees in 20 min.
-Kenneth: The restriction enzymes are deactivated and excluded when you purify the samples by gel electroforesis followed by [[Team:Copenhagen/Protocol#Gel Extraction Protocol|gel extraction]]. This approach exclude the restrictionenzymes and other contaminations, but has aswell a down side - you loose some of the DNA.
-Freeze until you need them. The BioBrick is now ready for ligation.
-'''Adapted from'''
-The BioBrick™ Assembly Manual from NEB and Ginkgo BioWorks
-http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf
-==Fourth Week==
-=== Gel Extraction Protocol===
-*After gelelectroforesis cut out the DNA bands (approx.. 1600 bp). Visualize by ultraviolet light (this does not damage the DNA as the Imaganizer does).
-*Weigh the gelpieces.
-'''Gel Extraction Protocol'''
-*Use 600 uL QG buffer pr. Gelpiece.
-*Incubate at 50°C until the pieces are meltet (approx. 10-15 min). Speed up the melting process by vortexing.
-*Add 1 gel volume (100mg ~100 uL) of Isopropanol (2-propanol) and mix.
-*Transfer the samples to QIAquick spin columns (max. 800 uL) and centrifuge for 1 min. For samples volumes of more than 800 uL simply load and spin again.
-*Discard flow-through.
-*Add 500 uL QG buffer and centrifuge for 1 min.
-*Discard the flow-through.
-*Add 750 uL PE buffer and centrifuge for 1 min. IF you are to use the DNA for blunt-end ligation or direct sequencing let the column stand for 2-5 min. before centrifugation.
-*Discard the flow-through
-*Spin again for 1 min and discard the flow-through.
-*Place the QIAquick column into an Eppendorff tube.
-*Eluate the DNA by adding 50 uL EB buffer. Remember to load the EB buffer in the center of the membrane. Let the column stand for 1 min. Centrifuge for 1 min.
-*Freeze the samples for later use.
-You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.
-===Digestion of Linearized Plasmid Backbones===
-This protocol is intended only for the preparation of the plasmid backbones (and NOT the expression vectors), before you can ligate it with your BioBrick.
-'''25 uL Enzyme Master Mix'''
-uL NEB Buffer 2
-,5 uL BSA
-,5 uL EcoRI
-,5 uL PstI
-,5 uL DpnI
-uL H2O
-* Add 4 uL linearized plasmid backbone (100 ng in total)
-* Add 4 uL of the Master Mix
-* Digest at 37 degrees 30 min/'''O/N at 4 degrees'''.
-* We used two different approaches:
-iGEM: Deactivate the restriction enzymes by incubating at 80 degrees in 20 min. You now have a plasmid concentration of 12,5 ng/uL
-Kenneth: Purify the plasmids by gel electrophoresis followed by [[Team:Copenhagen/Protocol#Gel Extraction Protocol|gel extraction]].
-* Freeze until you need them. The plasmids are now ready for ligation.
-===Ligation Protocol===
-How to ligate a BioBrick into a plasmid backbone: Use 3 times insert (BioBrick) to 1 time plasmid (3:1) in molar amounts. In calculating the amounts of DNA consider the length of the BioBrick and the plasmid, see example below. Use 25-30 ng plasmid.
-Note: iGEM are ligating eqimolar amounts of insert and plasmid as the only one in the world.
-Following the two different approaches of digestion, where you obtain different concentrations of the DNA, use either iGEM or Kenneths procedure for ligation. See below.
-Use eppendorf tubes for the ligation, so you can add the competent cells directly into the tubes.
-''Example:''
-''pSB1C3: 3000 bp''
-''CYP79B1: 1600 bp''
-''3000/1600 ≈ 2 (molar ratio)''
-''25 ng/2= 12,5 ng (25 ng and 12,5 ng are equivalent molar amounts in grams)''
-''In this procedure we use 3 times excess of insert (B1):''
-''12,5 ng *3= 37,5 ng B1''
-'''iGEM'''
-uL digested plasmid (12,5 ng/uL)
-x uL digested BioBrick (10 ng/uL)
-uL T4 Ligase buffer
-,5 uL T4 DNA Ligase
-Add water to a final volume of 10 uL
-'''Our iGEM way'''
-x ul digested plasmid (25 ng)
-x ul digested BioBrick (~40 ng if the insert is half the size than the plasmid)
-ul T4 Ligase buffer
-,5 ul T4 DNA ligase
-Add water to a final volume of 10 ul
-'''Ligation O/N at 4 degrees'''
-'''Kenneth'''
-If you wish to use Kenneths digestion approach, you have to determine the concentrations of the BioBrick and the plasmid by NanoDrop.
-x uL gel extracted plasmid (25-30 ng)
-x uL gel extracted BioBrick
-uL T4 ligase buffer
-,5 uL T4 DNA ligase
-Add water to a final volume of 20 uL
-Ligate at room temperature for 10 min/'''Ligation O/N at 4 degrees'''
-Keep on ice until transformation.
-To ensure that the ligation has succeded make at least two different control saples:
-'''C1'''
-uL T4 ligase buffer
-,5 uL digested plasmid
-,5 uL H2O
-'''C2'''
-uL T4 ligase buffer
-,5 uL digested plasmid
-,5 uL T4 ligase
-uL H2O
-Transform the controls as if they were a real transformation containing a BioBrick.
-===Transformation Protocol for pSB1C3===
-The only exeption to the procedure is an incubation step after adding the LB medium. Incubation for 1 hr. at 37 degrees while shaking.
-Plate it on agar plates with chloramphenicol.
-===PCR of BioBrick in pSB1C3===
-uL HF buffer
-,4 uL 10 mM dNTP
-uL 10 pmol/uL primer A
-uL 10 pmol/uL primer B
-,2 uL x7 polymerase
-Add water uptil 20 uL
-Use a pipette tip to collect cells from the plate, and transfer it to the PCR mix. End by scrabing the tip against the inner surface of the PCR tube.
-'''PCR Program'''
-sec - 98°C
-sec - 68°C (The annealing temperature must never be higher than the extension temperature)
-sec - 72°C
-∞      - 12°C
-cycles
-Note: The annealing temperature is determined on the basis of the sequence of the primer. Use [https://www.finnzymes.fi/tm_determination.html the calculator]. Remember that the BioBrick has been shortened in the double digest, so it misses 8 bases. The primer still has theese 8 bases that don't anneal, and they should be excluded in the calculation. The annealing temperature must never be higher than the extension temperature
-(because of the length of the primers the annealing temperature calculated will probably be higher than the extension temperature, and should therefore be lowered)
-Note: The extension time should be 30 sek/kb
-==Fifth  Week==
-===Standard Assembly===
-* Double Digestion
-* Ligation
-* Transformation
-== Eighth Week==
-===Expression & Purification in BL21===
-'''Growing the cells'''
-Day 1:
-* Book the centrifuges for day 3.
-* Prepare starter culture in 5 mL LB + 5 uL antibiotics (amp) and grow O/N at 37 degrees.
-* Prepare TB medium for use the next day
- '''TB formula - for final 1L medium (incl. 100 mL buffer)'''
-,00 g Yeast Extract
-,00 g Tryptone
-mL 99% Glycetol
- H2O uptil 900 mL
- ''' Buffer (200 mL)''' 34 mL 1M KH2PO4
-mL 1M K2HPO4
- Add water uptil 200 mL
- The pH should end up around 7,2-7,4
-* Autoclave both the medium and the buffer.
-* Prepare 1 M 5-aminolevulinic acid hydrochloride.
-Day 2:
-* Inoculate 200 mL TB (180 mL medium + 20 mL buffer) with the 5 mL starter culture from yesterday. Grow the cells at 37°C while shaking until reaching ~OD600=0,5 (2-4 h.)
-* Take a sample of 1 mL for the SDS-PAGE assay.
- '''Sample preparation for the SDS-PAGE assay:'''
- * Dilute the samples (with water) until reaching ~OD600=0,5.
- * Spin down the cells for 5 min at 20000 G.
- * Discard the supernatant and resuspend the cells in 50 uL SDS buffer.
- '''The SDS buffer is very toxic - wear glows and work in the fume hood'''
-* Move the shaking flask to the 28°C shaker and induse the cells with '''steril''' 1 mM IPTG (inducing the promoter) and 1 mM 5-aminolevulinic acid hydrochloride (precursor of the HEM-group in p450).
-*Take further samples of 1 mL for the SDS-PAGE assay at 1, 2 & 4 hours.
-*The flask has to remain at 28°C O/N
-*Prepare Standard Buffer
- '''Standard Buffer'''
-mM Tricine pH 7,9
-mM NaCl
- Add 2mM DTT and 5mM EDTA on the day of use
-Day 3:
-*Take out the last sample for the SDS-PAGE assay.
-</p>
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Team:Copenhagen/Notebook

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Revision as of 20:15, 25 June 2012

Notebook