Quint Lab:transient transformation and expression

{| cellspacing="2px" cellpadding="0" border="0" style="padding: 0px; width: 750px; color: #000000; background-color: #ffffff;"
 * -valign="top"
 * width=300px style="padding: 5px; background-color: #ffffff; border: 2px solid #F8B603;" |

transient transformation and expression
note: we use this protocol for transient transformation of Nicotiana benthamiana leaves


 * 1) streak out Agrobacterium from –80°C and let grow over night on selective medium @ 30°C
 * 2) grow 2 ml over night culture in selective medium in glass tubes @ 30°C
 * 3) spin down in 2 ml tubes (4 min; 10000 rpm)
 * 4) remove SN
 * 5) resuspend in infiltration medium (appr. ml)
 * 6) dilute to an OD of 1.0 (for protein expression (WB, microscopy…)
 * 7) mix with P19 strain (silencing inhibitor strain) 1:1 → incubate for 1 h or more
 * 8) infiltrate leaves (normally lower side) with a syringe, label infiltrated area with edding
 * 9) (24) – 48 (better) hpi: collect leaf material with Korkbohrer

infiltration medium:
 * 10 mM MES pH5.3 – 5.5 	(stock 1 M → for 100 ml = 1 ml)
 * 10 mM MgCl2			(stock 1 M → for 100 ml = 1 ml)
 * 150 µg/ml Acetosyringone (in DMF) (stock 150 mg/ml → for 100 ml = 100 µl)