Team:Fatih Turkey/ecolitransformation
From 2011.igem.org
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<a href="https://2011.igem.org/Team:Fatih_Turkey/Human_Practice">Human Practice</a> | <a href="https://2011.igem.org/Team:Fatih_Turkey/Human_Practice">Human Practice</a> | ||
<ul> | <ul> | ||
- | <li><a href="https://2011.igem.org/Team:Fatih_Turkey/Sporocide"> | + | <li><a href="https://2011.igem.org/Team:Fatih_Turkey/Sporocide">Sporicide</a></li> |
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/iGEM_for_7_to_77">iGEM for 7 to 77</a></li> | <li><a href="https://2011.igem.org/Team:Fatih_Turkey/iGEM_for_7_to_77">iGEM for 7 to 77</a></li> | ||
<li><a href="https://2011.igem.org/Team:Fatih_Turkey/game">Game</a></li> | <li><a href="https://2011.igem.org/Team:Fatih_Turkey/game">Game</a></li> | ||
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<li><a | <li><a | ||
href="https://2011.igem.org/Team:Fatih_Turkey/Safety">Safety</a></li> | href="https://2011.igem.org/Team:Fatih_Turkey/Safety">Safety</a></li> | ||
+ | <li><a href="https://2011.igem.org/Team:Fatih_Turkey/collaboration">Collaboration</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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- | < | + | |
+ | <b><span class="style2">E.coli TOP10 Transformation </span> </b> | ||
<p>MATERIALS </p> | <p>MATERIALS </p> | ||
<p>· Heat block</p> | <p>· Heat block</p> | ||
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<p>- Autoclave the bottle.</p> | <p>- Autoclave the bottle.</p> | ||
<p>- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)</p> | <p>- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)</p> | ||
- | < | + | <b><span class="style5"> Transformation</span></b> |
<p>- Aseptic conditions prepared (70% EtOH, Bunsen burner etc.) </p> | <p>- Aseptic conditions prepared (70% EtOH, Bunsen burner etc.) </p> | ||
<p>- Place 500 uL LB in epp into heat block(42˚C).</p> | <p>- Place 500 uL LB in epp into heat block(42˚C).</p> |
Latest revision as of 19:52, 28 October 2011
MATERIALS
· Heat block
· Incubator with shaker
· Competent cell
· Lb broth
· Lb agar with antibiotic
· Parafilm
· Sterile MiliQ dH2O
· Ice
· 0,5 and 1,5 epp
· Spreader
SOLUTIONS
LB Agar Preparation
- Add 200 mL of dH2O to a graduated cyclindar.
- Transfer dH2O into glass bottle.
- Add 7 gr of LB-agar powder
- Autoclave the bottle.
- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)
- Pour the plates .
LB Broth Preparation
- Add 200 mL of dH2O to a graduated cyclindar.
- Transfer dH2O into glass bottle.
- Add 4 gr of LB powder
- Autoclave the bottle.
- After cooling, add 200 uL antibiotic (The LB agar solution should be cool enough not to damage to antibiotic)
Transformation- Aseptic conditions prepared (70% EtOH, Bunsen burner etc.)
- Place 500 uL LB in epp into heat block(42˚C).
- Thaw 50 uL competent cells on ice.
- Add 1 uL plasmid into the competent cell epp and spin for few sec.
- Incubate for 45 min on ice.
- Incubate at 42˚C for 80 sec in heat block.
- Incubate for 5 min on ice.
- Complete to 500 uL with pre-heated LB (42˚C).
-.Epp s adhered with tape to horizontal on shaker.
- Incubate at 37 C for 1 h at 240 rpm.
- Spread 125 uL from each tube on agar plates with suitable antibiotic.
- Incubate plates at 37˚C not longer than 12-14 h.