Rensselaer/7 August 2008
From 2008.igem.org
(New page: '''Promoter Group''' '''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 - ...) |
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'''Promoter Group''' | '''Promoter Group''' | ||
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+ | '''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''' | '''Ligation of Fe promoter in mRFP plasmid''' | ||
+ | |||
j61002 = 3002 bp | j61002 = 3002 bp | ||
+ | |||
Fe promoter insert = 1044 bp | Fe promoter insert = 1044 bp | ||
+ | |||
Fe promoter vector (j61002 - tet - mRFP) = 2252 bp | Fe promoter vector (j61002 - tet - mRFP) = 2252 bp | ||
+ | |||
mRFP vector (j61002 - tet) = 2952 bp | mRFP vector (j61002 - tet) = 2952 bp | ||
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''Recalculated [Fe promoter insert]'' = 25 ng/ul x (1044/(1044+2252)) = 8.5 ng/ul | ''Recalculated [Fe promoter insert]'' = 25 ng/ul x (1044/(1044+2252)) = 8.5 ng/ul | ||
- | 100 ng vector ( | + | ''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 | 3x insert (8.5 ng/ul): ((3)(1044/2952)(100))/8.5 = 12.5 ul | ||
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==> ''EtOH precipitation'' | ==> ''EtOH precipitation'' | ||
- | In 1.5 ml tube combine | + | 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 1.6 ul NaOAc pH 4.8 | ||
- | Add 1 ul glycogen. | + | Add 1 ul glycogen (acts as DNA carrier to help precipitation). |
Add 55.8 ul 100% EtOH. | Add 55.8 ul 100% EtOH. | ||
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Microfuge max speed 4C 10'. | Microfuge max speed 4C 10'. | ||
- | Discard supernatant | + | Discard supernatant. Wash pellet with 75% EtOH |
+ | |||
+ | Resuspend DNA pellet in 12.5 ul H2O. | ||
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DNA+H2O 12.5 ul | DNA+H2O 12.5 ul | ||
+ | |||
Ligation Buffer 1.5 ul | Ligation Buffer 1.5 ul | ||
+ | |||
T4 DNA Ligase 1 ul | T4 DNA Ligase 1 ul | ||
Incubate ON @ 15C | Incubate ON @ 15C |
Latest revision as of 19:56, 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