Susan's fast protocols

Cloning
PCR
Dilute whatever 100pmol/ul primers 5x. (I usually do 20x to 5uM and add 3ul)
10X buffer 5
25mM MgCl2 3
dNTP 5
DMSO 2.5
100x BSA 0.5
Template 1
5' primer 1
3' primer 1
KOD 1
Water
Total 50ul

PCR cycle
95C 2min
95C 30s
55C for plasmid template, 45C for genomic 30s
65C or 72C for elongation 1s/kb
Repeat 20x for plasmid template, 25x for genomic
65C or 72C for 5min
15C forever

Gel purify
Look using higher wavelength to not denature DNA
Digestion for an hour, more for vector
CIP vector
Gel purify vector, purify insert
Run gel 1ul vector, 1ul PCR product to find out quantity
Ligate 1 vector: 10 product in 20ul (control and ligation)15min RT

Incubate 10ul with 100ul competent cells on ice 5min
Heat shock 42C 1min
Ice 1 min
Recover (optional) 15min at 37C.
Spin down and resuspend in 100ul media
Plate and incubate O/N

Pick clones into 2ml 2YT each.
Miniprep in 5-6h.
Digest 20min
Run gel
Set up large prep.

Western blot
Harvest 1/3 confluent plate into eppie tube
Spin 5k 3min
Add ~40ul 4x loading dye
Boil 2min
Load 5ul marker, 8ul samples
Pour buffer between gels, halfway outside gels
Run 25mA/gel, 1kV, ~35min till blue dye reach bottom

Wet membrane with methanol
Soak filter papers in transfer buffer
Assemble filter papers, membrane, gel, filter papers.
Wet membrane to slide gel easily.
Ensure no bubbles.
Run 20V 48min

Block with 5% milk
Make boat with parafilm, incubate 1ul primary antibody in 1ml 5% milk, membrane face down, 1h rocking RT
Wash 3x 5min each
Incubate secondary antibody 1ul in 10ml 30min RT
Wash 3x 5min each

Mix ECL, 1ml A + 25ul B
Spread on membrane with pipette tip on Saran wrap
Incubate 1min
Dry membrane with Kimmies

Develop film (cut film in half and label where film was positioned)