result from 4/26 - competent cells - negative control
no colonies on any plates → fine
RT-PCR of medicago Aux/IAAs
use cDNAs from 4/25 and dilute 1:10 → use 1 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
1
dH2O
13.7
82.2
total
20
35 cycles → 1
primers:
AC138130_5-F/R → 981 bp
AC175828_16-F/R → 810 bp
AC148405_5-F/R → 642 bp
AC158504_5-F/R → 882 bp
AC146866_11-F/R → 1044 bp
AC152423_24-F/R → 627 bp
MtAOC1-F/R → 758 bp
gel 1% LE:
row1
lane
sample
1
MtAOC1 - petiole2
2
MtAOC1 - stem2
3
MtAOC1 - roots4
4
MtAOC1 - leaves2
5
MtAOC1 - flowers1
6
AC138130_5 - petiole2
7
AC138130_5 - stem2
8
AC138130_5 - roots4
9
AC138130_5 - leaves2
10
AC138130_5 - flowers1
11
AC175828_16 - petiole2
12
AC175828_16 - stem2
13
AC175828_16 - roots4
14
AC175828_16 - leaves2
15
AC175828_16 - flowers1
row2
lane
sample
1
AC148405_5 - petiole2
2
AC148405_5 - stem2
3
AC148405_5 - roots4
4
AC148405_5 - leaves2
5
AC148405_5 - flowers1
6
AC158504_5 - petiole2
7
AC158504_5 - stem2
8
AC158504_5 - roots4
9
AC158504_5 - leaves2
10
AC158504_5 - flowers1
11
AC146866_11 - petiole2
12
AC146866_11 - stem2
13
AC146866_11 - roots4
14
AC146866_11 - leaves2
15
AC146866_11 - flowers1
row3
lane
sample
1
AC152423_24 - petiole2
2
AC152423_24 - stem2
3
AC152423_24 - roots4
4
AC152423_24 - leaves2
5
AC152423_24 - flowers1
6
λ HindIII/EcoRI
7
MtAOC1 - leaves2
8
AC138130_5 - leaves2
9
AC175828_16 - leaves2
10
AC148405_5 - leaves2
11
AC158504_5 - leaves2
12
AC146866_11 - leaves2
13
AC152423_24 - leaves2
14
15
results:
AOC control looks fine, only transcripts in roots seem underrepresented → use different internal standard → b-actin
AC138130_5 PCR failed → repeat, if fails again make new primers
AC175828_16 looks fine, expressed everywhere, somewhat weaker in roots and leaves, but second band in petioles and roots → maybe homologs not known from draft sequence → (i) increase Ta or (ii) make new primers
AC148405_5 looks bad, smaller band seems to have the correct size → make new primers
AC158504_5 looks fine, weakest expression in roots (but might be due to lower amount of cDNA, see 1.)
AC146866_11 looks ok, but two bands (upper band has the correct size → (i) increase Ta or (ii) make new primers
AC152423_24 looks fine, only expressed in petioles and stem → repeat PCR to make sure amplification was fine in the other tissues
Medicago
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