Exeter/28 August 2013

NanoDrop

We collected NanoDrop data of previously miniprepped, defrosted DNA. The NanoDrop machine was zero-ed using the elution buffer from the gel extraction kit used.

• ompC A - 20.1 ng/µl
• ompC B - 9.3ng/µl
• [http://parts.igem.org/Part:BBa_B0034?title=Part:BBa_B0034 B0034] - 26.7ng/µl
• RFP - 198ng/µl

The concentration for ompF B was too low, so we continued on to the digestion with only ompF A. The concentration for RFP was too high, so we diluted it.

• RFP (dilution) - 50.4ng/µl

Digestion

We pipetted the following volumes into four 0.6 ml tubes, keeping all enzymes and buffers on ice. We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.

The tubes were incubated at 25^oC for 30 minutes and then 80^oC for 20 minutes.

Ligation

The ligation was carried out using two methods: equal volumes and equal moles. To calculate the equimolar volumes, we used the following data:

The ompF, B0034, AMP plasmid, and RFP were from the earlier digestions. We pippetted the following volumes into four 0.6ml tubes:

We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.

The tubes were incubated at 16^oC for 30 minutes and then 80^oC for 20 minutes.

Gel of Digestion

While carrying out the ligation, we ran a gel of our digested parts.

Take me back to the notebook.