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

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repeat (from 4/29) RT-PCR of medicago Aux/IAAs

  • use cDNAs from 4/25 and dilute 1:10 → use 2 µl of this dilution as template for PCR
  • 20 µl ExTaq Rx:
component vol 1x vol 6x
ExTaq 0.1 0.6
10x buffer 2 12
dNTPs [2.5mM] 1.6 9.6
P-F/R 1.6 9.6
template 2
dH2O 12.7 86.2
total 20

primers:
AC138130_5-F/R → 981 bp → [2] (Ta=53), but only 30 and 33 cycles
AC148405_5-F/R neu → 348 bp →[1] (Ta=50), 35 cycles
AC158504_5-F/R → 882 bp → [1] (Ta=50), but only 30 and 33 cycles
AC146866_11-F/R → 1044 bp → [3] + [4] (Ta=55 bzw. 57), 35 cycles
β-ACTIN F/R → ? bp → [1] (Ta=50), 30 + 33 + 35 cycles

  • gel 1% LE:


row1

2008-5-6-row1.jpg
lane sample
1 β-ACTIN - petiole2 - 30 cycles
2 β-ACTIN - stem2 - 30 cycles
3 β-ACTIN - roots4 - 30 cycles
4 β-ACTIN - leaves2 - 30 cycles
5 β-ACTIN - flowers1 - 30 cycles
6 β-ACTIN - petiole2 - 33 cycles
7 β-ACTIN - stem2 - 33 cycles
8 β-ACTIN - roots4 - 33 cycles
9 β-ACTIN - leaves2 - 33 cycles
10 β-ACTIN - flowers1 - 33 cycles
11 λ HIII/ERI marker


row2

2008-5-6-row2.jpg
lane sample
1 β-ACTIN - petiole2 - 35 cycles
2 β-ACTIN - stem2 - 35 cycles
3 β-ACTIN - roots4 - 35 cycles
4 β-ACTIN - leaves2 - 35 cycles
5 β-ACTIN - flowers1 - 35 cycles
6 AC148405_5 - petiole2 - 35 cycles
7 AC148405_5 - stem2 - 35 cycles
8 AC148405_5 - roots4 - 35 cycles
9 AC148405_5 - leaves2 - 35 cycles
10 AC148405_5 - flowers1 - 35 cycles
11 λ HIII/ERI marker


row3

2008-5-6-row3.jpg
lane sample
1 AC138130_5 - petiole2 - 30 cycles
2 AC138130_5 - stem2 - 30 cycles
3 AC138130_5 - roots4 - 30 cycles
4 AC138130_5 - leaves2 - 30 cycles
5 AC138130_5 - flowers1 - 30 cycles
6 AC138130_5 - petiole2 - 33 cycles
7 AC138130_5 - stem2 - 33 cycles
8 AC138130_5 - roots4 - 33 cycles
9 AC138130_5 - leaves2 - 33 cycles
10 AC138130_5 - flowers1 - 33 cycles
11 λ HIII/ERI marker


row4

2008-5-6-row4.jpg
lane sample
1 AC158504_5 - petiole2 - 30 cycles
2 AC158504_5 - stem2 - 30 cycles
3 AC158504_5 - roots4 - 30 cycles
4 AC158504_5 - leaves2 - 30 cycles
5 AC158504_5 - flowers1 - 30 cycles
6 AC158504_5 - petiole2 - 33 cycles
7 AC158504_5 - stem2 - 33 cycles
8 AC158504_5 - roots4 - 33 cycles
9 AC158504_5 - leaves2 - 33 cycles
10 AC158504_5 - flowers1 - 33 cycles
11 λ HIII/ERI marker


row5

2008-5-6-row5.jpg
lane sample
1 AC148866_11 - petiole2 - Ta=55°C
2 AC148866_11 - stem2 - Ta=55°C
3 AC148866_11 - roots4 - Ta=55°C
4 AC148866_11 - leaves2 - Ta=55°C
5 AC148866_11 - flowers1 - Ta=55°C
6 AC148866_11 - petiole2 - Ta=57°C
7 AC148866_11 - stem2 - Ta=57°C
8 AC148866_11 - roots4 - Ta=57°C
9 AC148866_11 - leaves2 - Ta=57°C
10 AC148866_11 - flowers1 - Ta=57°C
11 λ HIII/ERI marker

results:

  1. β-ACTIN: weak band only at 35 cycles → new primers or different gene as internal control?
  2. AC148405_5: new primers failed also → leave out this gene
  3. AC138130_5: Ta 53°C and 33 cycles seems fine → final conditions: 33 cycles, Ta=53°C
  4. AC158504_5: 30 cycles ok → try once more with Ta=53°C
  5. AC146866_11: fine → final conditions: 35 cycles, Ta=57°C

summary:

  • need to establish internal control
  • AC138130_5: fine → 33 cycles, Ta=53°C
  • AC175828_16: fine → 35 cycles, Ta=53°C
  • AC148405_5: cancelled
  • AC158504_5: 30 cycles ok, but increase Ta to 53°C
  • AC146866_11: fine → 35 cycles, Ta=57°C
  • AC152423_24: fine → 35 cycles, Ta=50°C