IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/03

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Ligation reaction for RBS and TesA

  • Independently digest the TesA (sample 1), RBS (sample 7) to combine with provided backbone (24KS EPKS)which has been cut at the E and P sites.

TesA

  • Digest 15 μL DNA with 3 μL Fast Digest buffer w/ dye, 1μL Xba1, 1 μL Pst1, and 10μL of water.

RBS

  • Digest 15 μL DNA with 3 μL Fast Digest buffer w/ dye, 1μL EcoR1, 1 μL Spe1, and 10μL of water.

Let the reactions digest for at least 15 minutes Actual time: about 20 minutes Ran in a 1% agarose gel made with SafeWhite

Result: Bands were faint

Repeated digest, but with the following procedure:

TesA

  • Digest 15 μL DNA with 3 μL Fast Digest buffer, 1μL Xba1, 1 μL Pst1, 5 μL loading dye, and 5 μL of water.

RBS

  • Digest 15 μL DNA with 3 μL Fast Digest buffer, 1μL EcoR1, 1 μL Spe1, 5 μL loading dye, and 5 μL of water.

Let the reactions digest for at least 15 minutes Actual time: about 20 minutes Ran in a 1% agarose gel made with SybrSafe

Result: Bands were inconclusive


Final Digests

Run digest overnight, then run in gel on 7/4

RBS with Spe1 and Pst1 TesA with Xba1 and Pst1 TesA with EcoR1 and Pst1