User:Marcel Quint/Notebook/medicago/2008/05/05

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Medicago
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

repeat (from 4/29) RT-PCR of medicago Aux/IAAs

 * use cDNAs from 4/25 and dilute 1:10 → use 2 µl of this dilution as template for PCR
 * 20 µl ExTaq Rx:

primers: AC138130_5-F/R	→ 981 bp → [2] (Ta=53), but only 30 and 33 cycles AC148405_5-F/R neu → 348 bp →[1] (Ta=50), 35 cycles AC158504_5-F/R → 882 bp → [1] (Ta=50), but only 30 and 33 cycles AC146866_11-F/R	→ 1044 bp → [3] + [4] (Ta=55 bzw. 57), 35 cycles β-ACTIN F/R → ? bp → [1] (Ta=50), 30 + 33 + 35 cycles


 * gel 1% LE:

row1

row2

row3

row4

row5 results:
 * 1) β-ACTIN: weak band only at 35 cycles → new primers or different gene as internal control?
 * 2) AC148405_5: new primers failed also → leave out this gene
 * 3) AC138130_5: Ta 53°C and 33 cycles seems fine → final conditions: 33 cycles, Ta=53°C
 * 4) AC158504_5: 30 cycles ok → try once more with Ta=53°C
 * 5) AC146866_11: fine → final conditions: 35 cycles, Ta=57°C

summary:

 * need to establish internal control
 * AC138130_5: fine → 33 cycles, Ta=53°C
 * AC175828_16: fine → 35 cycles, Ta=53°C
 * AC148405_5: cancelled
 * AC158504_5: 30 cycles ok, but increase Ta to 53°C
 * AC146866_11: fine → 35 cycles, Ta=57°C
 * AC152423_24: fine → 35 cycles, Ta=50°C


 * }