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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/DNA Kit
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<p class="texte">Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Transformation Efficiency Kit. | <p class="texte">Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Transformation Efficiency Kit. | ||
| - | To use the DNA in the Distribution Kit, follow these instructions: <br> | + | To use the DNA in the Distribution Kit, follow these instructions: <br><br> |
| - | Note: <i>There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul </i><br> | + | Note: <i>There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul </i><br></p> |
| - | 1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.< | + | <p class="list"> |
| - | 2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.< | + | <ul> |
| - | 3. Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.< | + | <li>1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.</li> |
| - | 4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.< | + | <li>2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.</li> |
| - | 5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.< | + | <li>3. Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.</li> |
| - | <i>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.</i> | + | <li>4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</li> |
| + | <li>5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.</li> | ||
| + | </ul></p> | ||
| + | |||
| + | <p class="texte"><i>* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.</i><br> | ||
Note: <i>There is not enough DNA in each well to perform anything but transformations.</i></p> | Note: <i>There is not enough DNA in each well to perform anything but transformations.</i></p> | ||
Revision as of 15:02, 2 September 2013
Notebook
Protocols
DNA Kit Plate Instructions (iGEM protocol)
Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Transformation Efficiency Kit.
To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul
- 1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
- 2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
- 3. Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
- 4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
- 5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations.
