User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/24

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χρόνος πέρασμα September 24th 2011

HydA CAI Optimization Control

ABSTRACT
  • Electrophoretic gels from the last PCRs I did this and this. I tried concentrating the products but nothing appeared on the gels.

  • Today I concentrated all the PCR products to see if once concentrated I could see any amplification (just as Paulina did here). The 12 PCR tubes from the PCR using Pfx Platinum Polymerase with the PCRx Enhancer Buffer at different concentrations as well as the 12 PCR tubes from the PCR using Taq Polymerase with a combination of primers were left into the SpeedVac for approximately 30 minutes at high temperature. Once most of the solvent got evaporated, I added 20 μl of HPLC water and left the tubes at 42°C during 20 minutes to dissolve the contents. I proceeded to run the samples into a 1% electrophoretic gel (130 V 35 m). 5 μl from the total of 20 μl from each reaction was used.


Dramirez 24-09-11 PCRsconcentrated Pfx.jpg
Well PCR product
1 250 bp Ladder
2 Empty
3 Empty
4 HydA NoOpt PCRx Enhancer Buffer 0.5x
5 HydA NoOpt PCRx Enhancer Buffer 1x
6 HydA NoOpt PCRx Enhancer Buffer 2x
7 HydA Opt Enhancer Buffer 0.5x 55°C
8 HydA Opt Enhancer Buffer 0.5x 61°C
9 HydA Opt Enhancer Buffer 0.5x 68°C
10 HydA Opt Enhancer Buffer 1x 55°C
11 HydA Opt Enhancer Buffer 1x 61°C
12 HydA Opt Enhancer Buffer 1x 68°C
13 HydA Opt Enhancer Buffer 2x 55°C
14 HydA Opt Enhancer Buffer 2x 61°C
15 HydA Opt Enhancer Buffer 2x 68°C



  • An amplification product can be seen for the wells #4 and #5 that correspond to the HydA wild-type. At higher temperatures this region is not amplified. No HydA Optimized amplification product can be seen.


Dramirez 24-09-11 PCRsconcentrated Taq.jpg


Well PCR product
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 HydAOpt 5'v2 Rv Suffix
6 HydA Opt 63°C Fw HydAOpt 5'v2 Rv Suffix
7 HydA Opt 72°C Fw HydAOpt 5'v2 Rv Suffix
8 HydA Opt 55°C Fw Preffix Rv HydAOpt 3'v2
9 HydA Opt 63°C Fw Preffix Rv HydAOpt 3'v2
10 HydA Opt 72°C Fw Preffix Rv HydAOpt 3'v2
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
14 Empty
15 Empty















  • No difference can be observed from the other gel (the one that isn't concentrated).