Team:Macquarie Australia/Protocols and Other Methods
From 2010.igem.org
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<ol><li>Briefly centrifuge the pGEM®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube. Calculate the amount of insert DNA required to give a 1:3 ratio of vector to insert </li> | <ol><li>Briefly centrifuge the pGEM®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube. Calculate the amount of insert DNA required to give a 1:3 ratio of vector to insert </li> | ||
<li>Set up ligation reactions as described below. Vortex the 2x Rapid Ligation Buffer vigorously before each use. Use 0.5mL tubes known to have low DNA-binding capacity. </li></ol> | <li>Set up ligation reactions as described below. Vortex the 2x Rapid Ligation Buffer vigorously before each use. Use 0.5mL tubes known to have low DNA-binding capacity. </li></ol> | ||
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+ | |||
+ | |||
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+ | |||
+ | <div align="center"> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Reagents</th> | ||
+ | <th>Standard Rxn</th> | ||
+ | <th>+ Control </th> | ||
+ | <th> Background control </th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>2 x Rapid Ligation Buffer, T4 DNA ligase</td> | ||
+ | <td>5uL</td> | ||
+ | <td>5uL</td> | ||
+ | <td>5uL</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>pGEM®-T Easy Vector (50ng)</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>PCR product</td> | ||
+ | <td>XuL</td> | ||
+ | <td>-</td> | ||
+ | <td>-</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Control insert DNA</td> | ||
+ | <td>-</td> | ||
+ | <td>2uL</td> | ||
+ | <td>-</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>T4 DNA ligase (3 Weiss units/uL)DNA</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | <td>1uL</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Deionized water to a final volume of</td> | ||
+ | <td>10uL</td> | ||
+ | <td>10uL</td> | ||
+ | <td>10uL</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </center> | ||
+ | <p> </div> |
Revision as of 09:39, 19 October 2010
Protocols and Other Methods Used
BioLine Isolate Genomic DNA Mini Kit:
- Pellet cell culture, decant, add 1mL CLS-TC
- Resuspend and transfer to Fastprep tube (BIO101)
- Fastprep 5.5g for 30 seconds, then spin @ 14,000 rpm for 5 minutes
- Recover 700uL then 700uL Binding Matrix
- Rotate for 5 minutes then spin for 1 minute
- Decant the supernatant and add 800uL SEWS then vortex
- Rotate for 5 minutes then spin for 1 minute
- Decant the supernatant then pulse spin and remove the last bit of ethanol
- Air dry for 5 minutes then resuspend in 200uL TE buffer
- Spin at 14,000 rpm for 3 minutes and recover 160uL
Promega Wizard® Plus SV Minipreps DNA Purification System:
- Centrifuge bacterial culture at 12,000 rpm for 5 minutes, pour off the supernatant and blot the inverted tube on a paper towel to remove excess media
- Add 250uL of Cell Resuspension Solution and completely resuspend the pellet by pippetting
- Add 250uL of Cell Lysis Solution and mix by inverting tube 4 times, incubate for no more than 5 minutes
- Add 10uL Alkaline Protease Solution and mix by inverting tube 4 times, incubate at room temperature for no more than 5 minutes
- Add 350uL of Neutralization Solution and immediately mix by inverting the tube 4 times
- Centrifuge the bacterial lysate at 14,000 rpm in a microcentrifuge for 10 minutes at room temperature
- Transfer cleared lysate to Spin Column by decanting without disturbing precipitate
- Centrifuge the supernatant at 14,000 rpm for 1 minute at room temperature, discard flowthrough
- Add 750uL of Column Wash Solution (diluted with 95% ethanol), to Spin Column
- Centrifuge at 14,000 rpm for 1 minute at room temperature, discard flow through
- Add 250uL of Column Wash Solution
- Centrifuge at 14,000 rpm for 2 minutes at room temperature
- Transfer the Spin Column to a new tube
- Elute the plasmid DNA by adding 100uL of Nuclease Free Water to the Spin Column
- Centrifuge at 14,000 rpm for 1 minute at room temperature
- Add 11uL 10x TE buffer for DNA storage at 4C
50uL of ssH20 added instead of 100uL for elution step
Promega pGEM®-T Easy Vector System:
(Ligation Using 2x Rapid Ligation Buffer)
- Briefly centrifuge the pGEM®-T Easy Vector and Control Insert DNA tubes to collect contents at the bottom of the tube. Calculate the amount of insert DNA required to give a 1:3 ratio of vector to insert
- Set up ligation reactions as described below. Vortex the 2x Rapid Ligation Buffer vigorously before each use. Use 0.5mL tubes known to have low DNA-binding capacity.
Reagents | Standard Rxn | + Control | Background control |
---|---|---|---|
2 x Rapid Ligation Buffer, T4 DNA ligase | 5uL | 5uL | 5uL |
pGEM®-T Easy Vector (50ng) | 1uL | 1uL | 1uL |
PCR product | XuL | - | - |
Control insert DNA | - | 2uL | - |
T4 DNA ligase (3 Weiss units/uL)DNA | 1uL | 1uL | 1uL |
Deionized water to a final volume of | 10uL | 10uL | 10uL |