User:Marcel Quint/Notebook/medicago/2008/05/29

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RT-PCR of medicago Aux/IAAs - internal standard test

  • use cDNAs from 4/25 and dilute 1:10 → use 2 µl of this dilution as template for PCR
  • repeat reaction from yesterday, but with less cycles
  • 25 µl DreamTaq Rx:
component vol 1x vol 6x
ExTaq 0.25 1.5
10x buffer 2.5 15
dNTPs [10mM] 2.5 15
P-F/R 2 12
template 2
dH2O 15.75 94.5
total 25


28/31 cycles → [1] for ActinMt F/R (Ta=50)
primers: ActinMt F/R → ? bp

  • gel 2% 3:1:

row1


results:
  • looks fine, but root sample seems to contain less cDNA
  • repeat root tissue and two other tissues for comparison with increasing amounts of root cDNA: 2-4-6-10 μL template
  • 29 cycles and Ta=50°C
  • gel 2% 3:1:

row2

lane sample
1 ActinMt F/R 28 cycles - petiole2
2 ActinMt F/R 28 cycles - stem2
3 ActinMt F/R 28 cycles - roots4
4 ActinMt F/R 28 cycles - leaves2
5 ActinMt F/R 28 cycles - flowers1
6 ActinMt F/R 31 cycles - petiole2
7 ActinMt F/R 31 cycles - stem2
8 ActinMt F/R 31 cycles - roots4
9 ActinMt F/R 31 cycles - leaves2
10 ActinMt F/R 31 cycles - flowers1
11 pUC19 MspI marker
lane sample
1 ActinMt F/R 29 cycles - petiole2 - 2μL
2 ActinMt F/R 29 cycles - roots4 - 2μL
3 ActinMt F/R 29 cycles - flowers1 - 2μL
4 ActinMt F/R 29 cycles - petiole2 - 2μL
5 ActinMt F/R 29 cycles - roots4 - 4μL
6 ActinMt F/R 29 cycles - flowers1 - 2μL
7 ActinMt F/R 29 cycles - petiole2 - 2μL
8 ActinMt F/R 29 cycles - roots4 - 6μL
9 ActinMt F/R 29 cycles - flowers1 - 2μL
10 ActinMt F/R 29 cycles - petiole2 - 2μL
11 ActinMt F/R 29 cycles - roots4 - 10μL
12 ActinMt F/R 29 cycles - flowers1 - 2μL
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