Team:Wash U/Protocol
From 2009.igem.org
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# The following 3 step thermocycle should be repeated up to 30 times: 1) Denaturation 94C for 15 seconds 2) Annealing 65C for 20 seconds 3) Extension (Elongation) 68C for 20 minutes. | # The following 3 step thermocycle should be repeated up to 30 times: 1) Denaturation 94C for 15 seconds 2) Annealing 65C for 20 seconds 3) Extension (Elongation) 68C for 20 minutes. | ||
# Finish cycling with 68C for 10 minutes. | # Finish cycling with 68C for 10 minutes. | ||
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:Note: Restriction sites E=EcoR1-HF; X=Xba1; S=Spe1; P=Pst1; M=Mixed Site | :Note: Restriction sites E=EcoR1-HF; X=Xba1; S=Spe1; P=Pst1; M=Mixed Site | ||
:To view the full BioBrick Manual procedures, please click [http://partsregistry.org/Help:Transformation_Protocol here]. | :To view the full BioBrick Manual procedures, please click [http://partsregistry.org/Help:Transformation_Protocol here]. | ||
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# Centrifuge at 12,000RPM for 1 minute to remove excess ethanol. | # Centrifuge at 12,000RPM for 1 minute to remove excess ethanol. | ||
# Elute DNA: Transfer column to fresh collection tube. Add 50uL of Elution Solution to the column. centrifuge at 12,000RPM for 1 minute. DNA is now present in the eluate and ready for immediate use or storage at -20C. | # Elute DNA: Transfer column to fresh collection tube. Add 50uL of Elution Solution to the column. centrifuge at 12,000RPM for 1 minute. DNA is now present in the eluate and ready for immediate use or storage at -20C. | ||
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# Incubate at room temperature for 10 minutes followed by 80C for 20 minutes. This will deactivate enzymes and improve transformation efficiency. | # Incubate at room temperature for 10 minutes followed by 80C for 20 minutes. This will deactivate enzymes and improve transformation efficiency. | ||
# The ligation step is now completed. You may wish to store the products at -20C or begin a transformation immediately. | # The ligation step is now completed. You may wish to store the products at -20C or begin a transformation immediately. | ||
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# Transfer the tubes to an incubator set at 80C for another 20 minutes. This step will deactivate the restriction enzymes. | # Transfer the tubes to an incubator set at 80C for another 20 minutes. This step will deactivate the restriction enzymes. | ||
# Digestion is now finished and products should be stored at -20C or proceed to Ligation. | # Digestion is now finished and products should be stored at -20C or proceed to Ligation. | ||
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# Let the mix stand for 10 minutes at room temperature before incubating at 80C for 20 minutes (deactivates enzymes). | # Let the mix stand for 10 minutes at room temperature before incubating at 80C for 20 minutes (deactivates enzymes). | ||
# Store the products at -20C until they are needed for a transformation. | # Store the products at -20C until they are needed for a transformation. | ||
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#Add another 3ul of ethidium bromide to the buffer near the positive electrode when halfway to completion | #Add another 3ul of ethidium bromide to the buffer near the positive electrode when halfway to completion | ||
#Switch power supply off when gel has run to completion | #Switch power supply off when gel has run to completion | ||
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:Note:Expected yields are 4 umol PCB per 6g Spirulina | :Note:Expected yields are 4 umol PCB per 6g Spirulina | ||
:Thanks to Dr. Clark Lagarias for providing this protocol. [[Team:Wash_U/Project#References|ref]] | :Thanks to Dr. Clark Lagarias for providing this protocol. [[Team:Wash_U/Project#References|ref]] | ||
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# Close & vortex to mix. | # Close & vortex to mix. | ||
# Put in -80 C. | # Put in -80 C. | ||
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:We found the Registry's protocol extremely helpful for designing primers and constructed most of our primers from this template. To visit the website, please click [http://partsregistry.org/Help:Primers/Design here]. | :We found the Registry's protocol extremely helpful for designing primers and constructed most of our primers from this template. To visit the website, please click [http://partsregistry.org/Help:Primers/Design here]. | ||
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# PCR is now complete and should be held at 4C. | # PCR is now complete and should be held at 4C. | ||
Note: any further streaking should be done with a sterile tip. | Note: any further streaking should be done with a sterile tip. | ||
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# Place the mini column in a clean collection tube and pipet 200 uL Buffer AE on the DNeasy membrane. Incubate at room temperature for 1 minute and then centrifuge for 1 minute at 12,000RPM. | # Place the mini column in a clean collection tube and pipet 200 uL Buffer AE on the DNeasy membrane. Incubate at room temperature for 1 minute and then centrifuge for 1 minute at 12,000RPM. | ||
# Repeat elution as described in step 9 with same collection tube to combine both elutes. | # Repeat elution as described in step 9 with same collection tube to combine both elutes. | ||
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* 2.5 microliters EtBr (Caution: EtBr is a known carcinogen) | * 2.5 microliters EtBr (Caution: EtBr is a known carcinogen) | ||
* Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig. | * Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig. | ||
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{{WashUbottom}} | {{WashUbottom}} |
Revision as of 18:18, 13 July 2009