K-L
Protocols 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.