"
Team:SDU-Denmark/labwork/Protocols/Trans
From 2012.igem.org
(Created page with "<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> <head> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <!-- //////////// Site Name ////...") |
|||
| (3 intermediate revisions not shown) | |||
| Line 261: | Line 261: | ||
<tr> | <tr> | ||
<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/revtrans">Reverse Transcriptase</a></td> | <td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/revtrans">Reverse Transcriptase</a></td> | ||
| - | <td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/mutagen"> | + | <td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/mutagen">Mutagenesis</a></td> |
<td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/pcrgelclean">PCR-,gel clean-up</a></td> | <td><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Protocols/pcrgelclean">PCR-,gel clean-up</a></td> | ||
| - | + | ||
</tr> | </tr> | ||
</table> | </table> | ||
| Line 273: | Line 273: | ||
<!-- /// velkomst ////--> | <!-- /// velkomst ////--> | ||
| - | <h1> Transformation of | + | <h1> Transformation of Competent TOP10 E. coli</i></h1> </b> |
<p> | <p> | ||
| - | 1) Start thawing the competent cells on ice for 30 min.</ | + | 1) Start thawing the competent cells on ice for 30 min.</br></br> |
| - | + | 2) Add 1 - 2 µL of the resuspended DNA to the 1.5ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.</br></br> | |
| - | 3) Close tube and incubate the cells on ice for 30 minutes.</ | + | 3) Close tube and incubate the cells on ice for 30 minutes.</br></br> |
| - | 4) Heat shock the cells by immersion in a preheated water bath at 42ºC for 60 seconds.</ | + | 4) Heat shock the cells by immersion in a preheated water bath at 42ºC for 60 seconds.</br></br> |
| - | 5) Incubate the cells on ice for 5 minutes.</ | + | 5) Incubate the cells on ice for 5 minutes.</br></br> |
| - | 6) Add 300 μl of S.O.C media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation</ | + | 6) Add 300 μl of S.O.C media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation</br></br> |
| - | 7) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.</ | + | 7) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.</br></br> |
| - | 8) Label petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation solution onto different dishes, and spread.</ | + | 8) Label petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation solution onto different dishes, and spread.</br></br> |
| - | 9) Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and untransformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is exerted by the bacteria, and inactivates the antibiotic outside of the bacteria.</ | + | 9) Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and untransformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is exerted by the bacteria, and inactivates the antibiotic outside of the bacteria.</br></br> |
10) You can pick a single colony, make a glycerol stock, grow up a liquid cell culture, make colony PCR and miniprep. | 10) You can pick a single colony, make a glycerol stock, grow up a liquid cell culture, make colony PCR and miniprep. | ||
Latest revision as of 21:10, 26 September 2012
| mRNA Isolation | PCR | Miniprep | Check Digest |
| 3A-Assembly | Colony-PCR | Transformation | Gel-electrophoresis |
| Reverse Transcriptase | Mutagenesis | PCR-,gel clean-up |
Transformation of Competent TOP10 E. coli
1) Start thawing the competent cells on ice for 30 min. 2) Add 1 - 2 µL of the resuspended DNA to the 1.5ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice. 3) Close tube and incubate the cells on ice for 30 minutes. 4) Heat shock the cells by immersion in a preheated water bath at 42ºC for 60 seconds. 5) Incubate the cells on ice for 5 minutes. 6) Add 300 μl of S.O.C media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation 7) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin. 8) Label petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation solution onto different dishes, and spread. 9) Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and untransformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is exerted by the bacteria, and inactivates the antibiotic outside of the bacteria. 10) You can pick a single colony, make a glycerol stock, grow up a liquid cell culture, make colony PCR and miniprep.
