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 it is necessary to get regions for Isothermal Assembly with PCR to make overlap between sequences.

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

1. 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

Week 2

9th -15th September=

Repeating successful PCRs for Ter-Back1 and try to get Ter-Back2 but both failed.

Giving more time in Initial Denaturalization (5:00 min) we got better results, even for difficult PCRs as HydEF1 and different annealing temperatures

Fig.3 Successful PCR for PFOR2 and HYDEF2, each three lanes. 1-Ladder 500bp 2-HydEF2 55°C 3-HydEF2 60°C 4-HydEF2 62°C 5-HydEF1 55°C 6-HydEF1 60°C 7-HydEF1 62°C 8-HydG 55°C 9-PFOR2 55°C 10-HydG 60°C 11-HydG 62°C 12-PFOR2 60°C 13-PFOR2 62°C

Band isolation for HydG and PFRO2 was performed, but there are remaining of DNA template.

Week 3

16th-24th September

A second band extraction was performed to get bona fide PCR products.

Ter-Back 2 was amplified to get more PCR product, and with the same instructions Ter-Back 1 appears slightly amplified. The difference lies in addition of DMSO, probably the given nature of Terminator need it.

Fig.4. Numbers (1) and (2) represents 54.7°C and 60.3 °C for annealing respectively. 1. Ladder 500bp 2. PFOR2 E.B.‡ 3. HydG E.B.‡ 4. Ter-Back1 DMSO (1) 5. Ter-Back1 DMSO (2) 6. Empty 7. Ter-Back2 DMSO (1) 8. Ter-Back2 DMSO (2) 9. Ter-Back1 Not DMSO (1) 10. Ter-Back1 Not DMSO (2) 11. Ter-Back2 Not DMSO (1) 12. Ter-Back2 Not DMSO (2)

See the description of this system here.