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Rensselaer/7 August 2008
From 2008.igem.org
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'''Promoter Group''' | '''Promoter Group''' | ||
| + | |||
| + | '''Phosphatase Vector Digest''' | ||
| + | |||
| + | H20 5 ul | ||
| + | |||
| + | DNA (35 ng/ul) 48 ul | ||
| + | |||
| + | 10x SAP Buffer 6 ul | ||
| + | |||
| + | SAP (3 U/ul) 1 ul | ||
| + | |||
| + | Total Volume 60 ul | ||
| + | |||
| + | Incubate @ 37C for 30' | ||
| + | |||
| + | Heat inactivate by incubating @ 65C 15' | ||
| + | |||
'''Ligation of Fe promoter in mRFP plasmid''' | '''Ligation of Fe promoter in mRFP plasmid''' | ||
Revision as of 17:55, 7 August 2008
Promoter Group
Phosphatase Vector Digest
H20 5 ul
DNA (35 ng/ul) 48 ul
10x SAP Buffer 6 ul
SAP (3 U/ul) 1 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
100 ng vector (35 ng/ul): 100/35 = 2.9 ul
3x insert (8.5 ng/ul): ((3)(1044/2952)(100))/8.5 = 12.5 ul
==> EtOH precipitation
In 1.5 ml tube combine 2.9 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
