Team:ETH Zurich/Materials
From 2013.igem.org
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- | <h1><b> | + | <h1><b> Preparation of stock solutions</b></h1> |
<b>Making LB media</b><br><br>Dissolve 12.5g of LuriaBroth in 500mL MilliQ H2O and autoclave<br><br> | <b>Making LB media</b><br><br>Dissolve 12.5g of LuriaBroth in 500mL MilliQ H2O and autoclave<br><br> | ||
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<b>Making LB-Agar</b><br><br> Dissolve 12.5g LuriaBroth in 500mL MIlliQ H2O,add 7.5g Agar.<br>Before use: Heat it inb the microwave and let it cool down to 50 degree celsius before adding Antibiotics.<br><br> | <b>Making LB-Agar</b><br><br> Dissolve 12.5g LuriaBroth in 500mL MIlliQ H2O,add 7.5g Agar.<br>Before use: Heat it inb the microwave and let it cool down to 50 degree celsius before adding Antibiotics.<br><br> | ||
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<b>Antibiotics stock solutions</b><br><br> Ampicillin (amp): 100mg/mL (1000X)<br>Dissolve 1g Ampicillin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.<br><br> | <b>Antibiotics stock solutions</b><br><br> Ampicillin (amp): 100mg/mL (1000X)<br>Dissolve 1g Ampicillin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.<br><br> | ||
Kanamycin (Kan): 50mg/mL (1000X)<br>Dissolve 0.5g Kanamycin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.<br><br> | Kanamycin (Kan): 50mg/mL (1000X)<br>Dissolve 0.5g Kanamycin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.<br><br> | ||
Chloramphenicol(cam): 34mg/mL (1000X)<br>Dissolve 0.34g Chloramphenicol in 10mL sterile H2O. Aliquot and store at -20 degree celsius.<br><br> | Chloramphenicol(cam): 34mg/mL (1000X)<br>Dissolve 0.34g Chloramphenicol in 10mL sterile H2O. Aliquot and store at -20 degree celsius.<br><br> | ||
All stock solutions have to be diluted 1:1000 times when used for Cultures/Plates<br><br> | All stock solutions have to be diluted 1:1000 times when used for Cultures/Plates<br><br> | ||
- | <b>Glycerol 20%</b><br><br>Mix 20mL of Glycerole 100% and 80mL of MilliQ H2O, autoclave<br><br> | + | <b>Glycerol 20%</b><br><br>Mix 20mL of Glycerole 100% and 80mL of MilliQ H2O, autoclave |
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+ | <h1><b>General cloning procedure</b></h1> | ||
+ | <b>Minipreps</b><br><br>Sigma Aldricht Miniprep kit : Elute in 50uL for higher plasmid concentrations.<br><br> | ||
+ | |||
<b>Restriction enzyme digest</b><br><br>50uL reaction Volume:<br>2ug of plasmid (max 45uL for low concentration minipreps)<br>5uL NEB buffer (which buffer for which enzzyme can be looked up on the NEB website)<br>add ddH2O up to 50uL<br>0.5/1uL of restriction enzyme (20000U/mL/10000U/mL)<br>Keep enzymes always on cooling block and don't take them out for too long.<br>Mix through pipetting <br><br>1h at 37 degree celsius<br>Heat inactivation : 20min at 65 degree celsius(not always necessary)<br><br>Dephosphorylation of backbones only (reduces plasmid slef ligation)<br>Add 1uL of Calf-intestine-phosphatase)<br>1h at 37 degree celsius<br>20 minutes heatinactivation at 65 degree celsius.<br><br>Add 10uL of loading buffer (6X)<br> Analysis on gel<br><br> | <b>Restriction enzyme digest</b><br><br>50uL reaction Volume:<br>2ug of plasmid (max 45uL for low concentration minipreps)<br>5uL NEB buffer (which buffer for which enzzyme can be looked up on the NEB website)<br>add ddH2O up to 50uL<br>0.5/1uL of restriction enzyme (20000U/mL/10000U/mL)<br>Keep enzymes always on cooling block and don't take them out for too long.<br>Mix through pipetting <br><br>1h at 37 degree celsius<br>Heat inactivation : 20min at 65 degree celsius(not always necessary)<br><br>Dephosphorylation of backbones only (reduces plasmid slef ligation)<br>Add 1uL of Calf-intestine-phosphatase)<br>1h at 37 degree celsius<br>20 minutes heatinactivation at 65 degree celsius.<br><br>Add 10uL of loading buffer (6X)<br> Analysis on gel<br><br> | ||
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+ | <b>Ligation</b><br><br>Sigma Aldricht Miniprep kit : Elute in 50uL for higher plasmid concentrations. | ||
+ | |||
[[File:StdAss.png|left|500px|thumb|<b>Figure 1. iGEM suffix insertion</b>]] | [[File:StdAss.png|left|500px|thumb|<b>Figure 1. iGEM suffix insertion</b>]] | ||
[[File:600px-3AAssembly.png|left|500px|thumb|<b>Figure 2. iGEM 3A Assembly</b>]] | [[File:600px-3AAssembly.png|left|500px|thumb|<b>Figure 2. iGEM 3A Assembly</b>]] | ||
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<br clear="all"/> | <br clear="all"/> | ||
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+ | <h1><b>Experimental procedures/b></h1> |
Revision as of 14:55, 30 August 2013
Preparation of stock solutions
Making LB media
Dissolve 12.5g of LuriaBroth in 500mL MilliQ H2O and autoclave
Making LB-Agar
Dissolve 12.5g LuriaBroth in 500mL MIlliQ H2O,add 7.5g Agar.
Before use: Heat it inb the microwave and let it cool down to 50 degree celsius before adding Antibiotics.
Antibiotics stock solutions
Ampicillin (amp): 100mg/mL (1000X)
Dissolve 1g Ampicillin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.
Kanamycin (Kan): 50mg/mL (1000X)
Dissolve 0.5g Kanamycin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.
Chloramphenicol(cam): 34mg/mL (1000X)
Dissolve 0.34g Chloramphenicol in 10mL sterile H2O. Aliquot and store at -20 degree celsius.
All stock solutions have to be diluted 1:1000 times when used for Cultures/Plates
Glycerol 20%
Mix 20mL of Glycerole 100% and 80mL of MilliQ H2O, autoclave
General cloning procedure
Minipreps
Sigma Aldricht Miniprep kit : Elute in 50uL for higher plasmid concentrations.
Restriction enzyme digest
50uL reaction Volume:
2ug of plasmid (max 45uL for low concentration minipreps)
5uL NEB buffer (which buffer for which enzzyme can be looked up on the NEB website)
add ddH2O up to 50uL
0.5/1uL of restriction enzyme (20000U/mL/10000U/mL)
Keep enzymes always on cooling block and don't take them out for too long.
Mix through pipetting
1h at 37 degree celsius
Heat inactivation : 20min at 65 degree celsius(not always necessary)
Dephosphorylation of backbones only (reduces plasmid slef ligation)
Add 1uL of Calf-intestine-phosphatase)
1h at 37 degree celsius
20 minutes heatinactivation at 65 degree celsius.
Add 10uL of loading buffer (6X)
Analysis on gel
Ligation
Sigma Aldricht Miniprep kit : Elute in 50uL for higher plasmid concentrations.