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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Medicago
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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result from 4/26 - competent cells - negative control

 * no colonies on any plates → fine

RT-PCR of medicago Aux/IAAs
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
 * use cDNAs from 4/25 and dilute 1:10 → use 1 µl of this dilution as template for PCR
 * 20 µl ExTaq Rx:


 * gel 1% LE:

row1

row2

row3
 * 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


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