Team:Warsaw/SyntheticCloning/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 12h, 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.

• 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

You can see our results on the following gels

You can see the bands after 2h and 4h of RCR on 5ul of the starting DNA, and after 2h and 4h of RCR on 10ul of the starting DNA. On the gel you can see the digestions of 5ul of the RCR reaction (the reaction has 30ul) by the EcoRI and PstI enzymes.

We took 2ul of the starting DNA and got enough of the DNA for downstream processing after 12 hours, first results are visible after 6h.