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Team:Alberta/Notebook/protocols/transformations
From 2010.igem.org
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| + | |||
| + | ==General Protocols== | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/invitro_biobyte_assembly | In Vitro BioByte Assembly]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/LB | LB Plates and Broth]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/transformations |Transformations]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/overnight |5mL Overnight ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/glycerol | Glycerol Stock ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/miniprep | Plasmid Miniprep ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/digest | Restriction Digest ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/vector_dephos | Vector Dephosphorylation]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/ligation | Ligation ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/agarose_gel | Agarose Gel Electrophoresis ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/gel_extraction | Gel Extraction ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/pcr | PCR]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/colony_pcr | Colony PCR ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/pcr_purification | PCR Purification ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/labelling | Sample Labelling Conventions]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/sequencing | Fluorescent Sequencing Reaction]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/primer_design | Primer Design]] | ||
| + | ----------------------------- | ||
| + | |||
| + | |||
| + | {{Team:Alberta/endMainContent}} | ||
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Latest revision as of 02:20, 27 October 2010
Transformations
Reagents:
- Competent DH5α cells (100uL per transformation)
- up to 5uL of plasmid you want to transform
- 800uL of LB (non-contaminated) per transformation
- Plate with correct antibiotics
Procedure:
- Chill a box of pipette tips at -4oC and put your reaction tubes on ice.
- Obtain DH5α cells from the –80oC freezer and thaw in palm. Store on ice for 10 minutes.
- Use a chilled tip to transfer 100 ul of the DH5α cells into each tube. Set aside one tube as a control.
- Pipet up to 5uL of the plasmid (up to 50ng per 100ul of competent cells, plasmid amount. Should not exceed 5% that of the competent cells) into each tube. Swirl.
- Store on ice for 30 minutes.
- Place tubes in incubator set to 42oC for EXACTLY 90 seconds.
- Return the tubes to ice for 2 minutes.
- Add 800uL of LB to each tube.
- Incubate at 37oC for 45 minutes, ensure that the tube has at least 2ml capacity, to properly aerate the cells.
- Spread cells on plate with appropriate antibiotic: plate between 50- 300ul each. Let dry.
- Place plates inverted in incubator at 37oC for 12-16 hours.
