Team:TU Darmstadt/Protocols/mutagenic PCR

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=== Working principle ===
=== Working principle ===
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[[File:Mutagene_pcr_plasmid_v2.png|200px|thumb|right|Figure 1.]]
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[[File:Mutagene_pcr_plasmid_v2.png|200px|thumb|right|'''Figure 1.'''A shematic representation of mutagenic PCR]]
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The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.
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The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants were selected by restriction digestion with a disered restriction enzyme.
===Primers===
===Primers===
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===Mixtures===
===Mixtures===
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===PCR Program===
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{| class="wikitable"
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!Reaction mix !! Volume/Mass
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|-
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|5x Phusion HF buffer || 5µL
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|-
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|dNTP´s || 1µL
 +
|-
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|DMSO || 1,5 µL
 +
|-
 +
|DNA || 5,25,50 ng
 +
|-
 +
|Primer fwd || 125 ng
 +
|-
 +
|Primer rev || 125 ng
 +
|-
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|Phusion-Polymerase || 0,5 µL
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|-
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|H<sub>2</sub>O || '''ad 50 µL'''
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|}
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===Themocycler program===
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# T= 98°C 1 min
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# T= 63°C 0,5 min
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# T= 72°C 3 min
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# go to 2 rep 15
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# T= 72°C 8 min
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# Hold 4°C

Latest revision as of 22:36, 26 September 2012

Contents

Mutagenic PCR

This site-directed mutagenesis is used to change particular base pairs in a piece of DNA.

Working principle

Figure 1.A shematic representation of mutagenic PCR

The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The copied gene contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants were selected by restriction digestion with a disered restriction enzyme.

Primers

primers designed for this PCR:

Mixtures

Reaction mix Volume/Mass
5x Phusion HF buffer 5µL
dNTP´s 1µL
DMSO 1,5 µL
DNA 5,25,50 ng
Primer fwd 125 ng
Primer rev 125 ng
Phusion-Polymerase 0,5 µL
H2O ad 50 µL

Themocycler program

  1. T= 98°C 1 min
  2. T= 63°C 0,5 min
  3. T= 72°C 3 min
  4. go to 2 rep 15
  5. T= 72°C 8 min
  6. Hold 4°C
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