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METHODS (Neuronal cultures (Day 2 (Wash (Sterile water, 2 mL water, 3x),…
METHODS
Neuronal cultures
Day 1
Coat wells with PLL
PLL + borate buffer
0.5 mg/mL PLL (1 mL + 19 mL BB)
Shake vigorously when added
Keep in incubator overnight
Add 1 mL of PLL into each well
Day 2
Wash
Sterile water
2 mL water
3x
Add plating media
1.5 mL
Leaving in incubator overnight
Day 3
Change media
Fresh 1.5 mL of plating media
Add neuronal cells
0.5 million per well
Leave in incubator overnight
Day 4
Change plating media to feeding media
Day 10???????
Add virus
Day 17????
Lyse
Straight into 1 x loading buffer
Aspirate media cells were in
250 uL of loading buffer
Scrape around
Pipette all liquid in the well up
95C for 10 mins
Cloning
PCR
Components
10X KOD Hot start buffer
25 mM MgSO4 --> 1.5 mM
2mM dNTPs --> 0.2 mM
Primers
Forward
TCGACTAGTCGCCACCATGCTGCCCGGTTTGGCA
APP and Kozak
Reverse
Myc + Stu I site
CACAGGCCTTTAATTCAAGTCCTCTTCAGA
Template DNA
APP Swedish mutation
KOD Hot start DNA pol
Agarose gels
in 0.5X TEA
0.8%
15 min 135 V
Restriction digest
Ligation
Plasmids
Purification
GeneJET gel extraction
GeneJET midiprep kit
Western Blotting
Gels
10% agarose
Run in lab running buffer
20 uL sample
3 uL ladder + 17 uL loading buffer
10 mins 100V, 50 mins 180V
Membrane transfers
PVDF membrane
Methanol
0.4 A 1 hr 10 min
Set up
Rack
Sponges
Filter paper
Blotting
Blocking
5% BSA
2g BSA + 40 mL PBST
6% Milk
2.4 g milk in 40 mL PBST
Antibodies
phospho-abs in BSA
Rest in milk
Table this
All 1 hour RT or overnight at 4C
Developing
LICOR odessy
ECLs
Left for 1 minute
HEK cultures
Making virus
Checking APP expression