Rensselaer/7 August 2008

Promoter Group

Phosphatase Vector Digest

H20                     4 ul

DNA (35 ng/ul)         48 ul

10x SAP Buffer          6 ul

SAP (3 U/ul)            2 ul

Total Volume           60 ul

Incubate @ 37C for 30'

Heat inactivate by incubating @ 65C 15'

Ligation of Fe promoter in mRFP plasmid

j61002 = 3002 bp

Fe promoter insert = 1044 bp

Fe promoter vector (j61002 - tet - mRFP) = 2252 bp

mRFP vector (j61002 - tet) = 2952 bp

Calculations

Recalculated [Fe promoter insert] = 25 ng/ul x (1044/(1044+2252)) = 8.5 ng/ul

Recalculated [mRFP vector] = ((35 ng/ul)(50 ul))/60 ul = 29 ng/ul

100 ng vector (29 ng/ul): 100/29 = 3.4 ul

3x insert (8.5 ng/ul): ((3)(1044/2952)(100))/8.5 = 12.5 ul

==> EtOH precipitation

In 1.5 ml tube combine 3.4 ul vector and 12.5 ul insert (total volume ~ 16 ul)

Add 1.6 ul NaOAc pH 4.8

Add 1 ul glycogen (acts as DNA carrier to help precipitation).

Add 55.8 ul 100% EtOH.

Dry ice 15'.

Microfuge max speed 4C 10'.

Discard supernatant. Wash pellet with 75% EtOH

Resuspend DNA pellet in 12.5 ul H2O.

Ligation Reaction

DNA+H2O         12.5 ul

Ligation Buffer  1.5 ul

T4 DNA Ligase      1 ul

Incubate ON @ 15C