Team:ETH Zurich/lab/protocols

From 2014.igem.org

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(DNA Purification by Centrifugation)
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According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
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According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
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===Transformation buffer 2 (TFB2)===
===Transformation buffer 2 (TFB2)===
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According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
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===Gibson Assembly reaction mixture===
===Gibson Assembly reaction mixture===
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Aliquots of 15 μL can be stored at -20 °C
Aliquots of 15 μL can be stored at -20 °C
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==Methods==
==Methods==
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According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
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===Preparation of DNA from iGEM kit===
===Preparation of DNA from iGEM kit===
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For confirmation all plasmids used from the iGEM kit were sequenced
For confirmation all plasmids used from the iGEM kit were sequenced
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===Transformation of competent ''E. coli''===
===Transformation of competent ''E. coli''===
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According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
According to [http://www.qiagen.com/resources/molecular-biology-methods/dna/ QIAGEN ''DNA protocols and applications'']
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===Plasmid preparation===
===Plasmid preparation===
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The [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html Sigma-Aldrich GenElute&trade; Plasmid Miniprep Kit] was used
The [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html Sigma-Aldrich GenElute&trade; Plasmid Miniprep Kit] was used
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===Preparation of samples for sequencing at Microsynth===
===Preparation of samples for sequencing at Microsynth===
Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).
Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).
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===PCR procol for phusion DNA polymerase===
===PCR procol for phusion DNA polymerase===
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| H<sub>2</sub>O || add to reach a total volume of 20 μL ||  add to reach a total volume of 50 μL
| H<sub>2</sub>O || add to reach a total volume of 20 μL ||  add to reach a total volume of 50 μL
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===Restriction Endonuclease Reaction (double digestion)===
===Restriction Endonuclease Reaction (double digestion)===
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Enzymes, buffers and protocol are from New England BioLabs
Enzymes, buffers and protocol are from New England BioLabs
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===DNA Purification by Centrifugation===
===DNA Purification by Centrifugation===
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According to Promega Wizard SV Gel and PCR Clean-Up System, Quick protocol
According to Promega Wizard SV Gel and PCR Clean-Up System, Quick protocol
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===Agarose gel electrophoresis===
===Agarose gel electrophoresis===
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#For a 5 cm x 6 cm gel add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
#For a 5 cm x 6 cm gel add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
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#Fill the samples and ladder in the wells
#Fill the samples and ladder in the wells
#Run the gel at 135 V
#Run the gel at 135 V
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===Preparation of cryostocks===
===Preparation of cryostocks===
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#Add 750 μL sterile glycerol (30%) to 750 μL bacteria culture in a screw top tube
#Add 750 μL sterile glycerol (30%) to 750 μL bacteria culture in a screw top tube
#Freeze the glycerol stock tube at -80 °C
#Freeze the glycerol stock tube at -80 °C
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===Site-directed mutagenesis===
===Site-directed mutagenesis===
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Protocol based on [http://2014.igem.org/wiki/index.php?title=Team:ETH_Zurich/lab/protocols&action=edit&section=19 QuikChange Site-Directed Mutagenesis]
Protocol based on [http://2014.igem.org/wiki/index.php?title=Team:ETH_Zurich/lab/protocols&action=edit&section=19 QuikChange Site-Directed Mutagenesis]
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===Gibson Assembly===
===Gibson Assembly===
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# Run the reaction for 30-60 min at 50 °C
# Run the reaction for 30-60 min at 50 °C
# Subsequently the reaction mixture (5 uL are enough) can be used directly to transform competent cells (75 uL)
# Subsequently the reaction mixture (5 uL are enough) can be used directly to transform competent cells (75 uL)
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===Preparation of alginate beads===
===Preparation of alginate beads===

Revision as of 09:12, 25 August 2014

iGEM ETH Zurich 2014