K-L
Lysis Buffer (miniprep)
5 ml final vol
0.2 M NaOH 100 µl of 10 M
1% SDS 250 µl of 20% SDS
ddH2O 4.65 ml
Loading Buffer (6X or 10X,
DNA)
6X:
0.25% Bromophenol blue
0.25% Xylene cynol FF
30% Glycerol
69.5% H2O
10X:
5 ml glycerol
40 mg Xylene cynol FF
40 mg Bromophenol blue
0.4 ml 0.5 M EDTA
QS to 10 ml with dd H2O
*ALSO SEE REDUCING BUFFER
LB-Ampicillin
-20 g LB
-Bacto-Agar 15 g (omit if only want
liquid broth.
-Add 1 L dH2O.
-Autoclave and let cool until the
erlermeyer flask can be picked up by
the bare hand.
-Add 1 ml of 100 mg/ml Amp
-Mix well and pour plates.
Ligation (3 mixes)
Mix 1 2 3 .
3 µl PQE 3 µl PQE 1 µl PQE
3 µl Insert 1 µl Insert 3 µl Insert
4 µl Lig. buffer 4 µl Lig. buffer 4 µl Lig. buffer
0.5 µl ATP (0.1 M) 0.5 µl ATP (0.1 M) 0.5 µl ATP (0.1 M)
8.5 µl H2O 10.5 µl H2O 10.5 µl H2O
Incubate @ 16°C o/n. Heat shock ligation into appropriate E. coli
strain for cloning.