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Team:Fatih Turkey/ecolitransformation
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| + | <p><span class="style2">E.coli TOP10 Transformation </span> </p> | ||
<p>MATERIALS </p> | <p>MATERIALS </p> | ||
<p>· Heat block</p> | <p>· Heat block</p> | ||
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<p>· 0,5 and 1,5 epp</p> | <p>· 0,5 and 1,5 epp</p> | ||
<p>· Spreader</p> | <p>· Spreader</p> | ||
| - | <p> | + | <p> </p> |
| + | <p>SOLUTIONS</p> | ||
<p><strong>LB Agar Preparation</strong></p> | <p><strong>LB Agar Preparation</strong></p> | ||
<p>- Add 200 mL of dH2O to a graduated cyclindar.</p> | <p>- Add 200 mL of dH2O to a graduated cyclindar.</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> | ||
| - | <p>< | + | <p><span class="style5"> Transformation</span></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> | ||
<p><strong>- </strong>Thaw 50 uL competent cells on ice.<strong> </strong></p> | <p><strong>- </strong>Thaw 50 uL competent cells on ice.<strong> </strong></p> | ||
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<p><strong>- </strong>Spread 125 uL from each tube on agar plates with suitable antibiotic.<strong> </strong></p> | <p><strong>- </strong>Spread 125 uL from each tube on agar plates with suitable antibiotic.<strong> </strong></p> | ||
<p><strong>- </strong>Incubate plates at 37˚C not longer than 12-14 h.</p> | <p><strong>- </strong>Incubate plates at 37˚C not longer than 12-14 h.</p> | ||
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</div> | </div> | ||
Revision as of 17:34, 27 October 2011
E.coli TOP10 Transformation
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.
Sponsors
