User:Marcel Quint/Notebook/medicago/2008/04/29

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repeat (from 4/28) 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

35 cycles → [1] for AC138130_5 and AC152423_24 (Ta=50) + [2] for AC175828_16 and AC146866_11 (Ta=53)
primers:
AC138130_5-F/R → 981 bp
AC175828_16-F/R → 810 bp
AC146866_11-F/R → 1044 bp
AC152423_24-F/R → 627 bp

  • gel 1% LE:

row1

2008-4-29-row1.jpg
lane sample
1 AC138130_5 - petiole2
2 AC138130_5 - stem2
3 AC138130_5 - roots4
4 AC138130_5 - leaves2
5 AC138130_5 - flowers1
6 AC152423_24 - petiole2
7 AC152423_24 - stem2
8 AC152423_24 - roots4
9 AC152423_24 - leaves2
10 AC152423_24 - flowers1
11 λ HIII/ERI marker

row2

2008-4-29-row2.jpg
lane sample
1 AC175828_16 - petiole2
2 AC175828_16 - stem2
3 AC175828_16 - roots4
4 AC175828_16 - leaves2
5 AC175828_16 - flowers1
6 AC146866_11 - petiole2
7 AC146866_11 - stem2
8 AC146866_11 - roots4
9 AC146866_11 - leaves2
10 AC146866_11 - flowers1
11 λ HIII/ERI marker

results:

  1. AC138130_5: ok, but increase Ta to 53°C + use less cycles
  2. AC152423_24: fine → final conditions: 35 cycles, Ta=50°C
  3. AC175826_16: fine → final conditions: 35 cycles, Ta=53°C
  4. AC146866_11: better, but still additional bands → increase Ta to 55 and 57°C

from yesterday:

  1. AC148405_5: make new primers
  2. AC158504_5: Ta=50°C ok, but use less cycles → try 32 and 29