Team:Chiba/Notebook/protocol
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+ | :::::<Font Size="7">'''''E''.coli Time Manager '''</Font> <html><a href="http://www.chiba-u.ac.jp/e/" target="_blank"><img src="https://static.igem.org/mediawiki/2009/5/5f/Logo_chiba-u.gif" align="right" width="250"></a></html> | ||
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+ | {| style="color:white;" cellpadding="2" cellspacing="0" border="0" width="100%" align="center" class="menu" |align="center" | ||
+ | !align="center"|[[Team:Chiba|Home]] | ||
+ | !align="center"|[[Team:Chiba/Team|The Team]] | ||
+ | !align="center"|[[Team:Chiba/Parts|Parts]] | ||
+ | !align="center"|[[Team:Chiba/Reference|Reference]] | ||
+ | !align="center"|[[Team:Chiba/Notebook|Notebook]] | ||
+ | !align="center"|[[Team:Chiba/Notebook/protocol|Protocols]] | ||
+ | !align="center"|[[Team:Chiba/Links|Links]] | ||
+ | !align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]] | ||
+ | !align="center"|[[Team:Chiba/Contact|Contact]] | ||
+ | |} | ||
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- | <table width="100%" border="1" cellpadding="1" cellspacing="0" align="center" bordercolor="#800000" align="center"> | + | <table class="calendar" width="100%" border="1" cellpadding="1" cellspacing="0" align="center" bordercolor="#800000" align="center"> |
<tr><td> | <tr><td> | ||
- | [[Team:Chiba| | + | '''[[Team:Chiba/Project|The Project]]''' |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project| | + | '''1,''' [[Team:Chiba/Project#Introduction|Introduction]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project | + | '''2,''' [[Team:Chiba/Project#Project_Design|Project Design]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project | + | '''3,''' [[Team:Chiba/Project#Experiments,_Results_&_Discussion|Experiments, Results & Discussion]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project | + | 3-1, [[Team:Chiba/Project#Making_LuxR_Mutants|Making LuxR Mutants]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project | + | 3-2, [[Team:Chiba/Project#Characterization_of_LuxR_Mutants|Characterization of LuxR Mutants]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/ | + | 3-3, [[Team:Chiba/Project#Demonstration|Demonstration]] |
</td></tr><tr><td> | </td></tr><tr><td> | ||
- | [[Team:Chiba/Project | + | '''5,''' [[Team:Chiba/Project#Conclusions|Conclusions]] |
</td></tr></table> | </td></tr></table> | ||
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__NOTOC__ | __NOTOC__ | ||
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== Contents == | == Contents == | ||
- | + | #[[Team:Chiba/Notebook/protocol#Transformation|Transformation (Using Zymo comp.)]] | |
- | + | #[[Team:Chiba/Notebook/protocol#DNA_Purification|DNA_Purification]] | |
- | + | ##[[Team:Chiba/Notebook/protocol#Sigma_prep|Sigma prep]] (Plasmid mini prep) | |
- | + | ##[[Team:Chiba/Notebook/protocol#Zymo_DNA_Cleam&Concentrator_Kit|Zymo DNA Cleam&Concentrator Kit]] | |
- | + | #[[Team:Chiba/Notebook/protocol#Agarose_gel_electrophoresis|Agarose gel electrophoresis]] | |
- | + | #[[Team:Chiba/Notebook/protocol#Digestion|Digestion]] | |
- | + | #[[Team:Chiba/Notebook/protocol#PCR|PCR]] | |
- | + | #[[Team:Chiba/Notebook/protocol#Gel_extract|Gel extract]] | |
- | + | #[[Team:Chiba/Notebook/protocol#Dephosphorylation_of_DNA|Dephosphorylation of DNA]] | |
- | + | #[[Team:Chiba/Notebook/protocol#Ligation|Ligation]] | |
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== Protocols == | == Protocols == | ||
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Day 1 night | Day 1 night | ||
- | *Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 | + | *Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 °C until OD is 0.4-0.6. |
Day 2 | Day 2 | ||
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+ | ===DNA Purification=== | ||
- | |||
====Sigma prep==== | ====Sigma prep==== | ||
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*Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA. | *Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA. | ||
+ | |||
===Agarose gel electrophoresis=== | ===Agarose gel electrophoresis=== | ||
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#Load it into agalose gel | #Load it into agalose gel | ||
#Run the gel at ~100 volts for 35 mins. | #Run the gel at ~100 volts for 35 mins. | ||
+ | |||
*Visualizing agarose gels | *Visualizing agarose gels | ||
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===Digestion=== | ===Digestion=== | ||
- | + | #Mix (in a PCR tube) | |
+ | ##plasmid DNA | ||
+ | ##buffer | ||
+ | ##Restriction Enzyme | ||
+ | ##NFW | ||
+ | #Incubate at 37ºC for 3h | ||
===PCR=== | ===PCR=== | ||
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===Gel extract=== | ===Gel extract=== | ||
+ | *'''Agalose Gel casting''' | ||
+ | #Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer | ||
+ | #Microwave until the agarose is fully melted | ||
+ | #Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid | ||
+ | #Remove comb | ||
+ | |||
+ | |||
+ | *'''Running agalose gel''' | ||
+ | #Load 5 μL prepared 1kbp ladder | ||
+ | #Mix DNA solution with loading dye(6x) and water | ||
+ | #Load it into agalose gel | ||
+ | #Run the gel at ~100 volts for 35 mins. | ||
+ | |||
+ | |||
+ | *'''Visualizing agarose gels''' | ||
+ | #Remove gel from gel box | ||
+ | #Soak the gel in ethidium bromide solution | ||
+ | #Let it 30 min. | ||
+ | #Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing. | ||
+ | #Print the picture. | ||
+ | #Remove gel and throw in trash | ||
+ | #Wipe down Trans-Illuminator if necessary. | ||
- | |||
- | + | *'''Extract''' | |
#Cut the agarose target band | #Cut the agarose target band | ||
#The chip of the gel into 2mL ADB buffer | #The chip of the gel into 2mL ADB buffer | ||
#Let it in 37 degree 30 min to solve the agarose gel. | #Let it in 37 degree 30 min to solve the agarose gel. | ||
- | # | + | #[[Team:Chiba/Notebook/protocol#Zymo_DNA_Cleam&Concentrator_Kit|Purify the DNA with Zymo DNA Cleam&Concentrator Kit]] |
- | + | ||
- | + | ||
===Dephosphorylation of DNA=== | ===Dephosphorylation of DNA=== | ||
- | SAP: Alkaline Phosphatase (Shrimp) | + | '''SAP: Alkaline Phosphatase (Shrimp)''' |
- | + | #Mix | |
- | + | ##DNA fragment 1~10 pmol | |
- | + | ##shrimp Alkaline Phosphatase (1~5 μ l) 1~5 U | |
- | + | ##10X SAP Buffer 5 μ l | |
- | + | ##Sterilized distilled water up to 50 μ l | |
+ | #Incubate at 37°C for 15~30 min. | ||
+ | #Incubate at 65°C for 15 min. (for inactivation by heat treatment) | ||
+ | #[[Team:Chiba/Notebook/protocol#Zymo_DNA_Cleam&Concentrator_Kit|Purify the DNA with Zymo DNA Cleam&Concentrator Kit]] | ||
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===Ligation=== | ===Ligation=== | ||
- | + | #Mix insert DNA with vector DNA. | |
+ | #Add 1u Invitrogen Ligase and Ligase-Buffer(x5). | ||
+ | #Store RT for 3h. | ||
- | + | ===Time Delay Test=== | |
#Transformed sender and receiver into E coli strains. | #Transformed sender and receiver into E coli strains. | ||
- | #Inoculated them independently in liquid media. Incubated at | + | #Inoculated them independently in liquid media. Incubated at 37°C 12h. |
- | #Inoculated again in Fresh liquid media upto about OD600=2 at | + | #Inoculated again in Fresh liquid media upto about OD600=2 at 37°C |
#Washed sender and receiver. | #Washed sender and receiver. | ||
#Mixed them. (Sender:Receiver=1000μL:1000μL) | #Mixed them. (Sender:Receiver=1000μL:1000μL) |
Latest revision as of 22:51, 21 October 2009
Home | The Team | Parts | Reference | Notebook | Protocols | Links | Acknowledgements | Contact |
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Contents
ProtocolsTransformationDay 1 morning
Day 1 night
Day 2
DNA PurificationSigma prepZymo DNA Cleam&Concentrator Kit
Agarose gel electrophoresis
Digestion
PCR
Start: 94 °C for 5 min. (melt) cycle: melt: 1 min. anneal : 30 sec. cycle end: extension:72 °C for 3.5 min. 25 cycles 72 °C for 10 min store: keep at 6 °C forever
Gel extract
Dephosphorylation of DNASAP: Alkaline Phosphatase (Shrimp)
Ligation
Time Delay Test
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