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Rensselaer/7 August 2008
From 2008.igem.org
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
