Lab Logbook - System Assembling.

Process overview

Week 1

2nd - 8th September After 2 months of delay DNA synthesis from Mr. Gene has arrived at 22:00 hrs. They are 6 syntheses HydA, PFOR1, PFOR2, HydEF1, HydEF2 and HydG. PFORs and HydEFs were synthesized separately because sequences are too long for synthesis.

1-With this arrival is necessary get regions for Isothermal Assembly with PCR to make overlap between sequences.

2-Trasnsform cells with HydA and PFOR1 to get plasmid and split a Periplasm Tag (PT) designed by us.

3- PCR for Terminator-backbone (Ter-Back) in Isothermal Assembly.

First attempt failed but not at all, just PFOR_Dos and hydG amplified and positive control, for that we need a troubleshooting for PCR. The same case was for Ter-Back . There is a slight amplification of HydEF1.

First attempt in troubleshooting for HydEFs failed, this time we use other Polymerase (Taq Platinum) and didn´t work. Changing annealing temperatures we had a possible way to get HydEF2.

Plasmid extraction of HydA and PFOR was made.

Digestion with NdeI to Split PT was done, but results are confusing, because self Ligation didn´t work.

In order to do a band extraction we need more PCR product to successful PCRs we amplify them, band extraction was polluted by template DNA.

There is a possible way to get HydEF2, is necessary check that and perform more PCRs for more product. Ter-Back 1 amplified but Ter-Back2 didn´t.

Fig.2. Successful PCR of Terminator 1. Lanes 1. Ladder 500bp 2. Ter-Back 1 3. Ter-Back 2 4.HydEF 1 5. HydEF 2