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
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:
- Actin F/R: ok, very short fragment,use less cycles
- ActinMt F/R: ok, very short fragment,use less cycles
- 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
|
Medicago
