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
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
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:
AC138130_5: ok, but increase Ta to 53°C + use less cycles
AC152423_24: fine → final conditions: 35 cycles, Ta=50°C
AC175826_16: fine → final conditions: 35 cycles, Ta=53°C
AC146866_11: better, but still additional bands → increase Ta to 55 and 57°C
from yesterday:
AC148405_5: make new primers
AC158504_5: Ta=50°C ok, but use less cycles → try 32 and 29
Medicago
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