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__july
07-04-2008
Assess Pool
We mixed all oligos together, except for the modified, the left and the right borders and the remainders:
Oligos modified with flourophor: 1. board:
r-3t8f
r-5t10e
r-5t12e
Oligos modified with NIP: 1. board:
r-7t6f
r-7t8f
r-7t10f
r-5t8f
r-3t10e
r-7t6e
3. board:
r7t22e
Left border: 1.board, 1.line
Right border: 3.board, 2.line
Remainders: 3.board, 3.line
07-06-2008
Assessment of cell-culture
-(DYT-Medium; ER 2738-cells)
50ml DYT-Medium
+50 TET (25ng/ml -> 1:1000)
+ pick ER 2738-cells (from -80 degree freezer)
-> shake at 37 degree over night
07-07-2008
1) Thin down overnight culture to OD~0.1
1:10 -> 0.66 = OD 660
5000µl = 0.66
x = 0.1 -> x = 758µl ~ 760µl in 50ml
We mixed 760µl cell culture with 50ml DYT and shaked both at 37°C; Inoculation after 95min
<p>2) Inoculation of cell culture with M13mp18 phages
~ 50ml cell culture
+ 5µl M13mp18 phages (from -80°C)
-> 4h shaking at 37°C
3) Phage precipitation
1) centrifuging of the phage-solution at 5000 x g for 20 min.
2) after decanting supernatant dessolve the pellet in 7ml (1/7 of the supernatant volume) PEG/NaCl
-> The precipitation occurs at 4°C over night
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