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Team:Copenhagen/Protocol
Contents |
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
10 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
1 μl of dNTP mix
ddH2O to a final volume of 50 μl
Then add
1 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.
1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction.
2. 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
1. Inoculate a single colony into 5mL Lb in 15mL falcon tube. Grow O/N at 37°C.
2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.
3. Shake at 37°C for 1.5-3hrs until OD600 = 0.4-0.8
Then…. 1. Put the cells on ice for 10 mins (keep cold form now on).
2. Collect the cells by centrifugation in the big centrifugue for 10 mins at 6krpm
3. Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
4. Incubate on ice x 20 mins
5. Centrifuge as in 2
6. Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol (from a 85% stock)
7. 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
1. 500 ml LB agar and 500 μL amphicilin 2. Pour on plates 3. Leave with the lid half on for 30 minutes at room temperature 4. Put in refrigerator until needed.
Transformations
Transformation of Ca++ competent cells
1. Put ~50μL of competent cells to prechilled microtubes. Wait 1 minute. Add 10μL of circular plasmid or all of a ligation reaction of plasmid DNA.
2. Incubate for 15 mins on ice.
3. Heat shock for 30 seconds at 42°C. Put back on ice.
4. Add 70 μL LB
4. Plate the whole lot in LBamp plates
5. Leave the plates at 37°C O/N
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
1. Pellet 5 ml bacterial overnight culture by centrifugation at 4500 rpm for 10 min at room temperature (20°C).
2. Resuspend pelleted bacterial cells in 500 μl Buffer P1 and transfer and divide in two microcentrifuge tubes.
3. Add 250 μl Buffer P2 to each tube and mix thoroughly by inverting the tube 4–6 times until the solution becomes blue. Do not allow the lysis reaction to proceed for more than 5 min.
4. Add to each 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. With LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60 s and discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB. Centrifuge for 60 s and discard the flow-through
8. 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.
9. Centrifuge for 1 min to remove residual wash buffer.
10. 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
1μl BSA (10x) diluted ca. 500ng Plasmid 1μl Restrictionenzyme 1μl 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
PCR Reaction
We add prefix and suffix to our newly made BioBricks with PCR
Ingredients
Water to a final volume of 50μl
10 μl 5x Pfusion HF buffer
1 μl 10mM dNTP
0,5μM Primer A
0,5μM Primer A
0,5 μl template (ca. 150 ng Template)
0,50 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
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
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.
2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
5. Discard flow-through. Place the QIAquick column back into the same tube.
6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.
8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
9. 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)
5 μl NEB buffer (Decide which buffer: [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp])
0,5 μl BSA (Not necessary if the enzymes are HF)
1 μl Restrictionsenzyme A (Decide which restriction enzymes: [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf])
1 μl Restrictionsenzyme B
Mix by flicking
Incubate for 30 min in 37°C
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 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
5 uL NEB Buffer 2
0,5 uL BSA
0,5 uL EcoRI
0,5 uL PstI
0,5 uL DpnI
18 uL H2O
- Add 4 uL linearized plasmid backbone (100 ng in total)
- Add 4 uL of the Master Mix
- Digest at 37 degrees in 30 min.
- 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 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
2 uL digested plasmid (12,5 ng/uL)
x uL digested BioBrick (10 ng/uL)
1 uL T4 Ligase buffer
0,5 uL T4 DNA Ligase
Add water to a final volume of 10 uL
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
2 uL T4 ligase buffer
0,5 uL T4 DNA ligase
Add water to a final volume of 20 uL
Ligate at room temperature for 10 min.
Keep on ice until transformation.
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.
