Rensselaer/7 August 2008

From 2008.igem.org

Revision as of 18:07, 7 August 2008 by Simeod2 (Talk | contribs)

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.

Add 55.8 ul 100% EtOH.

Dry ice 15'.

Microfuge max speed 4C 10'.

Discard supernatant and 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