Team:Chiba/Notebook/protocol
From 2009.igem.org
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!align="center"|[[Team:Chiba/Contact|Contact]] | !align="center"|[[Team:Chiba/Contact|Contact]] | ||
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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#Transformation|Transformation (Using Zymo comp.)]] | ||
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+ | ===DNA Purification=== | ||
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====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. | ||
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===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. | ||
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*Visualizing agarose gels | *Visualizing agarose gels | ||
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READ:[[Team:Chiba/Notebook/protocol#Zymo_DNA_Cleam&Concentrator_Kit|Zymo_DNA_Cleam&Concentrator_Kit]] | READ:[[Team:Chiba/Notebook/protocol#Zymo_DNA_Cleam&Concentrator_Kit|Zymo_DNA_Cleam&Concentrator_Kit]] | ||
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===Dephosphorylation of DNA=== | ===Dephosphorylation of DNA=== | ||
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===Ligation=== | ===Ligation=== | ||
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+ | ===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 37°C 12h. | #Inoculated them independently in liquid media. Incubated at 37°C 12h. |
Revision as of 02:56, 29 August 2009
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Contents
ProtocolsTransformationDay 1 morning
Day 1 night
Day 2
DNA PurificationSigma prepZymo DNA Cleam&Concentrator Kit
Agarose gel electrophoresis
DigestionPCR
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 extractREAD:Agarose_gel_electrophoresis
READ:Zymo_DNA_Cleam&Concentrator_Kit
Dephosphorylation of DNASAP: Alkaline Phosphatase (Shrimp)
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
READ:Zymo cleam
Phosphorylation of DNALigationTime Delay Test
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