User:Marcel Quint/Notebook/medicago/2008/04/28

From OpenWetWare
< User:Marcel Quint‎ | Notebook‎ | medicago‎ | 2008‎ | 04
Jump to: navigation, search
Owwnotebook icon.png Medicago <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>      </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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

2008-4-28-row1.jpg
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

2008-4-28-row2.jpg
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

2008-4-28-row3.jpg
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
LambdaHIII-ERI.jpg
  • results:
  1. AOC control looks fine, only transcripts in roots seem underrepresented → use different internal standard → b-actin
  2. AC138130_5 PCR failed → repeat, if fails again make new primers
  3. 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
  4. AC148405_5 looks bad, smaller band seems to have the correct size → make new primers
  5. AC158504_5 looks fine, weakest expression in roots (but might be due to lower amount of cDNA, see 1.)
  6. AC146866_11 looks ok, but two bands (upper band has the correct size → (i) increase Ta or (ii) make new primers
  7. AC152423_24 looks fine, only expressed in petioles and stem → repeat PCR to make sure amplification was fine in the other tissues