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Team:UNAM-Genomics Mexico/Notebook/HydA
From 2011.igem.org
Lab Logbook - HydA.
Week 1
8th- 14 th August Design of the primers that are to be used to amplify the CDS regions of the optimized and wild-type HydA
Week 2
15th -21st August
Week 3
22nd- 28nd August Transformation of the bioparts J04500 and B0015 into DH5α E. coli
Week 4
29th August-4th September Plasmid extraction of J04500 and and B0015. Restrictions for both bioparts PstI-Spe for J04500, and EcoRI-XbaI B0015, and Gel extraction
Week 5
Week September 5 – September 11 PCR for two hydrogenases (HydA), the optimized and the wild-type, that I need to assemble the construction we need in order to test the codon optimization we performed on the coding regions of our genetic cirtuit; in theory, Rhizobium etli CFN42 growth shouldn't be affected by the insertion of the plasmids carrying our system and the expression of these, as the usage of codons of these sequences is similar to those sequences that are naturally highly transcribed (i.e. housekeeping genes). I used a Taq platinum polymerase from Invitrogen for the procedure. I had previously isolated the plasmid that carries the wild-type HydA gene, and given that it was relatively concentrated, I diluted it in a 1:100 manner adding HPLC water to prepare it as the PCR template. Also, I took 1 μl from the stock of HydA-Ferredoxin fusion (just as it came synthesized) and diluted it 1:20 with HPLC water, in order to have it as the optimized HydA template. For these, Paulina had already prepared the primers at the adequate concentration from the stock, they are at 5 μmol/μl.
The PCR reactions were prepared as follows:
| Reactants | Volume |
|---|---|
| H20 | 34 μl |
| PCR Buffer 10x | 5 μl |
| 0.4mM dNTPs mixture | 4 μl |
| 50mM MgCl2 | 1 μl |
| Primer forward 5μmol | 2 μl |
| Primer reverse 5μmol | 2 μl |
| DNA template | 1.5 μl |
| Taq platinum Pol | 0.5 μl |
| Total | 50μl |
I prepared two reactions for each desired amplification; 2 for HydAOpt and 2 for HydA NonOpt.
- Incubation 3 minutes at 94°C
- 30 Cycles
- Denature: 30 seconds at 94°C
- Anneal: 55°C for 30 seconds
- Extend: 1:55 minutes at 72°C
- Post-Incubation: 2 minutes at 72°C
Second attempt for HydA Opt was tried but it failed.
Week 6
12th -18th September Unsuccessful PCRs for HydA Opt and NonOpt with 55°C, 63°C, 72°C annealing temperatures for each one. 19th-25th September Now using Pfx Polymerase, with standard and PCRx enchancer buffer, PCRs were perforemed.. It didn´t amplified. Was needed a troubleshooting with different primer set. Even with this, there are no amplifications. Positive control amplified. We don´t really know what is happening.
