User:Marcel Quint/Notebook/medicago/2008/05/28

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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
  • use AC138130_5 as positive control for PCR rx
  • 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

35 cycles → [2] for Actin F/R (Ta=53) + [1] for ActinMt F/R, β-Act F/R, and AC138130_5 (Ta=50)
primers:
Actin F/R → ? bp
ActinMt F/R → ? bp
β-Act F/R → ? bp
AC138130_5-F/R → 981 bp

  • gel 1% LE:

row1

lane sample
1 Actin F/R - petiole2
2 Actin F/R - stem2
3 Actin F/R - roots4
4 Actin F/R - leaves2
5 Actin F/R - flowers1
6 ActinMt F/R - petiole2
7 ActinMt F/R - stem2
8 ActinMt F/R - roots4
9 ActinMt F/R - leaves2
10 ActinMt F/R - flowers1
11 λ HIII/ERI marker


row2

lane sample
1 β-Act F/R - petiole2
2 β-Act F/R - stem2
3 β-Act F/R - roots4
4 β-Act F/R - leaves2
5 β-Act F/R - flowers1
6 AC138130_5 - petiole2
7 AC138130_5 - stem2
8 AC138130_5 - roots4
9 AC138130_5 - leaves2
10 AC138130_5 - flowers1
11 λ HIII/ERI marker

results:

  1. Actin F/R: ok, very short fragment,use less cycles
  2. ActinMt F/R: ok, very short fragment,use less cycles
  3. β-Act F/R: root does not come up, discard

taken together: use ActinMt F/R as internal standard with Ta=50°C, try 30 cycles

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