Team:EPF-Lausanne/Protocols/Site-specific mutagenesis

From 2011.igem.org

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(→Primer Design)
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The easiest way to design primers is to copy the wild-type sequence into the sequence, then "upload and translate" it. A little clarification to the provided documentation: when the sequence is uploaded, check boxes appear below a set of radio-buttons. They list the amino acids, translated from the provided sequence. Check up to seven positions, then choose what substitutions to operate using the drop-down menus ''above''.
The easiest way to design primers is to copy the wild-type sequence into the sequence, then "upload and translate" it. A little clarification to the provided documentation: when the sequence is uploaded, check boxes appear below a set of radio-buttons. They list the amino acids, translated from the provided sequence. Check up to seven positions, then choose what substitutions to operate using the drop-down menus ''above''.
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our primers are then ordered from Invitrogen. Upon arrival, they must be diluted to XXX
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our primers are then ordered from Invitrogen. Upon arrival, they must be diluted ideally to 100 ng/µl. The primers we order contain too much DNA for the volume of the tube at that concentration, so we dilute them instead to '''1 µg/µl'''.
== Culture media preparation ==
== Culture media preparation ==
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* '''NZY+ Broth''' (autoclaved)
* '''NZY+ Broth''' (autoclaved)
* '''TE Buffer''' which can just be taken from a miniprep or gel extraction kit.
* '''TE Buffer''' which can just be taken from a miniprep or gel extraction kit.
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The agar broth is used to pour plates, as described in the [[Team:EPF-Lausanne/Protocols/Agar_Plates|agar plates protocol]].
== Mutation reaction ==
== Mutation reaction ==
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To be continued, once the primers are received...
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The mutation reaction creates the mutant strand. It is carried out like a PCR, in a thermal cycler. Mix the following reagents, for each '''50 µl''' reaction volume:
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{|
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|+ Reagents for the mutation reactions
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! Qty || Reagent
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|-
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| 5 µl || 10x reaction buffer
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|-
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| 0.5 µl || Template DNA ''(or adjust to obtain 10-100 ng)''
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|-
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| 1.25 µl || Sense primer at 100 ng / µl ''(or adjust to obtain 125 ng)''
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|-
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| 1.25 µl || Antisense primer at 100 ng / µl ''(or adjust to obtain 125 ng)''
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|-
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| 1 µl || dNTP
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|-
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| 1.5 µl || Quicksolution
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|-
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| 1 µl || Quickchange Enzyme
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|-
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| 38.5 µl || ddH20 ''(or adjust to obtain 50 µl total)''
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|}
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All reagents (but the water) are included in the mutagenesis kit.
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=== Control Reaction ===
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The control reaction reveals the efficacy of plasmid mutations. It mutates a pWhitescript plasmid into a pBluescript plasmid. Competent cells subsequently transformed with the pBluescript plasmid express the beta-galactosidase gene, which turns them blue in presence of IPTG and X-gal.
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Mix the following reagents:
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{|
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|+ Reagents for 50 µl control reaction
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! Qty || Reagent
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|-
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| 5 µl || Reaction buffer
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|-
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| 5 µl || pWhitescript
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|-
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| 1.25 µl || Control primer #1
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|-
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| 1.25 µl || Control primer #2
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|-
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| 1 µl || dNTP
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|-
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| 1.5 µl || Quicksolution
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|-
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| 34 µl || ddH20
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|}
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=== Thermal Cycles ===
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Run both reactions with the heat cycles listed below. The control reaction needs a 2'30" extension step.
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{|
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|+ Heat cycles for mutagenesis plasmid copy
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! Segment || Cycles || Temp [°C] || Time
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|-
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| 1 || 1 || 95 || 2 min
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|-
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| 2 || 18 || 95 || 20 s
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|-
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| || || 60 || 10 s
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|-
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| || || 68 || 30 s / kb plasmid length
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|-
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| 3 || 1 || 68 || 5 min
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|}
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{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 07:45, 27 July 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"