User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/22

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χρόνος πέρασμα September 22th 2011

HydA CAI Optimization Control

  • PCR of the optimized HydA CDS using Taq Polymerase, troubleshooting using a combination of primers.

  • Today I tried again to amplify the synthesized and optimized coding region of HydA but now with another procedure. I used a Taq Polymerase from Alta Enzymes (in order to not to waste other more expensive enzymes). Following our advisor Fernando Montaño's advise, I now proceeded to test if the oligonucleotides I sent synthesize are ok. These are the combinations of primers I tested:

Forward primer - Reverse primer
Biobrick Preffix with Biobrick Suffix
Biobrick Preffix with HydAOpt 3'v2
HydAOpt 5'v2 with Biobrick Suffix
HydAOpt 5'v2 with HydAOpt 3'v2

  • The first pair is suppose to be a positive control, as the plasmid were the CDS of the optimized HydA contains the Standard Biobrick Preffix and Suffix. The second and third pair of primers test either the 5'v2 or 3'v2 I designed individually. The last pair of primers is the one I've been trying to make work. I also tried every combination of primers in a temperature gradient for the annealing step in the PCR, this goes from 55°C to 72°C. The PCR reactions were performed as follows:

Reactant Volume
H20 56.5 μl
10x Taq Polymerase Buffer 10 μl
8mM dNTP Mixture 2 μl
25mM MgCl2 6 μl
Primer forward 5μmol 10 μl
Primer reverse 5μmol 10 μl
DNA template (diluted 1:20) 2 μl
Taq Polymerase 0.5 μl
Total 100 μl
  • I also increased the dNTPs concentration according to this [1].
  • Incubation 3 minutes at 94°C
  • 30 Cycles
    • Denature 94°C 45 seconds
    • Anneal 55°C - 72°C 30 seconds
    • Extend 72°C 2 minutes
  • After the 30 cycles, 10 minutes at 72°C
  • After the PCR, I ran a 1% agarose electrophoretic gel to see if the amplifications were succesful. 130 V 35 minutes. 5 μl from each PCR tube was used to load the wells.

Well PCR reaction
1 250 bp Ladder
2 HydA Opt 55°C Fw Preffix Rv Suffix
3 HydA Opt 63°C Fw Preffix Rv Suffix
4 HydA Opt 72°C Fw Preffix Rv Suffix
5 HydA Opt 55°C Fw Preffix Rv HydAOpt 3'v2
6 HydA Opt 63°C Fw Preffix Rv HydAOpt 3'v2
7 HydA Opt 72°C Fw Preffix Rv HydAOpt 3'v2
8 HydA Opt 55°C Fw HydAOpt 5'v2 Rv Suffix
9 HydA Opt 63°C Fw HydAOpt 5'v2 Rv Suffix
10 HydA Opt 72°C Fw HydAOpt 5'v2 Rv Suffix
11 HydA Opt 55°C Fw HydAOpt 5'v2 Rv HydAOpt 3'v2
12 HydA Opt 63°C Fw HydAOpt 5'v2 Rv HydAOpt 3'v2
13 HydA Opt 72°C Fw HydAOpt 5'v2 Rv HydAOpt 3'v2

Dramirez PCR HydA Taq primercombination.jpg

  • As it can be seen, there is no clear amplification product, though something can be seen (well #12) that happens to be of the size of the expected amplification product using the primers HydAOpt 5'v2 and HydAOpt 3'v2 (that is, ~1.8kb). Another clearer band appears at well #9, but it is of size approximately ~650 bp, no idea what this should be. Anyway, the positive controls weren't succesful, this gives insight that maybe the plasmid itself is the one that's giving me problems.