User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/05

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χρόνος πέρασμα September 5th 2011

HydA CAI Optimization Control

  • PCR of the CDS of the optimized and wild-type HydA using Taq platinum from Invitrogen.

  • Today I make a PCR from the 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

  • After the PCR, I ran a 1% agarose electrophoretic gel to see if they did amplified. 130 V 35 minutes.

Well PCR reaction
1 1kb Ladder
2 HydA NonOpt 1
3 HydA NonOpt 2
4 HydA Opt 1
5 HydA Opt 2

Dramirez PCR taqplatinum HydAOpt HydANonOpt.jpg

  • As it can be seen, there was no amplification for the optimized HydA, and fortunately, there was for the wild-type. Perhaps the primers that amplify for the optimized HydA do not behave the same as the other pair; maybe adding something (DMSO) to stabilize the hybridization and to contend with the high GC content might help.