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Team:Warsaw/SyntheticCloning/Biobricks
From 2011.igem.org
(Difference between revisions)
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<div class="note">Rolling circle amplification of the BioBricks</div> | <div class="note">Rolling circle amplification of the BioBricks</div> | ||
<div>Did you know that it is possible get BioBrick DNA ready for cloning in just two hours. We didn't, but we figured out that we could use phi29 rolling circle amplification instead of transforming DNA into bacteria. First we got 2ul of the BioBrick DNA from the distribution, and did the rolling circle amplification. It worked fine after 12 h, but it was not fast enough for us. Fortunately RCR heavily depends on the amount of starting DNA. Therefore we took 5ul of the DNA in water straight from the distribution and obtain satisfactory results in 2 hours.See our gels. </div> | <div>Did you know that it is possible get BioBrick DNA ready for cloning in just two hours. We didn't, but we figured out that we could use phi29 rolling circle amplification instead of transforming DNA into bacteria. First we got 2ul of the BioBrick DNA from the distribution, and did the rolling circle amplification. It worked fine after 12 h, but it was not fast enough for us. Fortunately RCR heavily depends on the amount of starting DNA. Therefore we took 5ul of the DNA in water straight from the distribution and obtain satisfactory results in 2 hours.See our gels. </div> | ||
| - | <div class="note">How can You get Biobricks from the distribution in 2 | + | <div class="note">How can You get Biobricks from the distribution in 2 hours</div> |
<div> | <div> | ||
<ul> | <ul> | ||
<li>1. Resuspend DNA from the distribution in 10ul RNAse free water</li> | <li>1. Resuspend DNA from the distribution in 10ul RNAse free water</li> | ||
| - | <li>2. Use some of it as a template in RCR reaction ( | + | <li>2. Use some of it as a template in RCR reaction (recommended 5ul)</li> |
<li>3. Set up the annealing mix as described:</li> | <li>3. Set up the annealing mix as described:</li> | ||
| + | <ul> | ||
<li>5ul template</li> | <li>5ul template</li> | ||
<li>1ul Phi29 polymerase buffer</li> | <li>1ul Phi29 polymerase buffer</li> | ||
<li>1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO</li> | <li>1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO</li> | ||
<li>RNAse free water to 10ul</li> | <li>RNAse free water to 10ul</li> | ||
| + | </ul> | ||
<li>4. Set up the annealing reaction:</li> | <li>4. Set up the annealing reaction:</li> | ||
| + | <ul> | ||
<li>heat mix to 95C</li> | <li>heat mix to 95C</li> | ||
<li>cool down to 95C, slowly 0.1C/s works</li> | <li>cool down to 95C, slowly 0.1C/s works</li> | ||
| - | <li> | + | </ul> |
| + | <li>5. to the annealing mix add :</li> | ||
| + | <ul> | ||
<li>1ul 10umol DNTPs - it is best to use RNAse free</li> | <li>1ul 10umol DNTPs - it is best to use RNAse free</li> | ||
<li>1ul phi29 polymerase from Epicentre</li> | <li>1ul phi29 polymerase from Epicentre</li> | ||
| Line 34: | Line 39: | ||
<li>1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual</li> | <li>1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual</li> | ||
<li>RNAse free water to 30ul final volume </li> | <li>RNAse free water to 30ul final volume </li> | ||
| - | <li> | + | </ul> |
| - | <li> | + | <li>6. Incubate at 30C for at least 2h</li> |
| + | <li>7. The product is ready for digestion. We used 5ul of the RCR product and performed digestions in 30ul</li> | ||
</ul> | </ul> | ||
You can see our results on the following gels | You can see our results on the following gels | ||
Revision as of 19:35, 20 September 2011
Getting Biobricks from the distribution in 2 hours
Rolling circle amplification of the BioBricks
Did you know that it is possible get BioBrick DNA ready for cloning in just two hours. We didn't, but we figured out that we could use phi29 rolling circle amplification instead of transforming DNA into bacteria. First we got 2ul of the BioBrick DNA from the distribution, and did the rolling circle amplification. It worked fine after 12 h, but it was not fast enough for us. Fortunately RCR heavily depends on the amount of starting DNA. Therefore we took 5ul of the DNA in water straight from the distribution and obtain satisfactory results in 2 hours.See our gels.
How can You get Biobricks from the distribution in 2 hours
- 1. Resuspend DNA from the distribution in 10ul RNAse free water
- 2. Use some of it as a template in RCR reaction (recommended 5ul)
- 3. Set up the annealing mix as described:
- 5ul template
- 1ul Phi29 polymerase buffer
- 1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO
- RNAse free water to 10ul
- 4. Set up the annealing reaction:
- heat mix to 95C
- cool down to 95C, slowly 0.1C/s works
- 5. to the annealing mix add :
- 1ul 10umol DNTPs - it is best to use RNAse free
- 1ul phi29 polymerase from Epicentre
- 2ul phi 29 buffer
- 1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual
- RNAse free water to 30ul final volume
- 6. Incubate at 30C for at least 2h
- 7. The product is ready for digestion. We used 5ul of the RCR product and performed digestions in 30ul
