IGEM:Harvard/2006/NES Notebook: Difference between revisions
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*Digestions - Vary enzyme concentration | *Digestions - Vary enzyme concentration | ||
- 8 ul Midiprep Insert DNA + .5 ul | - 8 ul Midiprep Insert DNA + .5 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 6ul H2O -> 45C 6H | ||
- 8 ul Midiprep Insert DNA + 1 ul | - 8 ul Midiprep Insert DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 5ul H2O-> 45C 6H | ||
- 8 ul Midiprep Insert DNA + 1 ul | - 8 ul Midiprep Insert DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 5ul H2O-> 37C 6H | ||
- 8 ul Midiprep Backbone DNA + .5 ul | - 8 ul Midiprep Backbone DNA + .5 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 6ul H2O-> 45C 6H | ||
- 8 ul Midiprep Backbone DNA + 1 ul | - 8 ul Midiprep Backbone DNA + 1 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 5ul H2O-> 45C 6H | ||
- 8 ul Midiprep Backbone DNA + 1 ul | - 8 ul Midiprep Backbone DNA + 1 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 5ul H2O-> 37C 6H | ||
Revision as of 11:22, 21 August 2006
To Order
- DMSO
- Sodium Thiosulfate
- Small, Medium, Large, X-large Nitrile gloves
Introductory Materials
New Reporters
Due to issues with T7 promoter, the following biobricks were rehydrated 6/14
Constitutive (IPTG-inducable?) GFP
Constitutive (IPTG?) RFP
Constitutive GFP using TetR promoter. No experience; might not work.
Constitutive (IPTG?) YFP. Apparently gives uneven YFP expression
Transformed with 2ul of DNA on 15ul bacteria; twice for each plasmid. Took 3 ul from each incubation of and added into two two mixed SOC cultures. Let's see what happens when LacI GFP, RFP, YFP are co-cultured.
One of each LacI plasmid type, GFP, RFP, YFP, LacI were cultured overnight on plates treated with 10mM IPTG.
Microscope Notes
Overlap between FITC and YGFP blocks prevents differentiation between YFP and GFP. RFP can be differentiated between YFP/GFP very well.
YFP/GFP fades ~3 hours after being taken out of the incubator after overnight incubation. RFP was still strong at +3h.
IPTG doesn't make an apparent difference in fluoresence.
Promoter Research
7/25/06 Made Glycerol cultures of
- J04430 (lac GFP)
- J04450 (lac RFP)
- I13522 (tet GFP)
We need to transfer these constructs onto pSB4A3
Ligation Assay
- Digestions - Vary enzyme concentration
- 8 ul Midiprep Insert DNA + .5 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 6ul H2O -> 45C 6H
- 8 ul Midiprep Insert DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 5ul H2O-> 45C 6H
- 8 ul Midiprep Insert DNA + 1 ul X+P enzyme to 25ul + 2.5ul Buffer + 2.5ul BSA + 5ul H2O-> 37C 6H
- 8 ul Midiprep Backbone DNA + .5 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 6ul H2O-> 45C 6H
- 8 ul Midiprep Backbone DNA + 1 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 5ul H2O-> 45C 6H
- 8 ul Midiprep Backbone DNA + 1 ul S+P enzyme to 25ul + 2.5ul Buffer #1 + 2.5ul BSA + 5ul H2O-> 37C 6H
- Phosphatase
- 25ul Digest + 1ul Phosphatase 1H at 45
- Run on Gel - Create purified and unpurified samples
Add 12 ul of each to the gel.
Chose the most successful digest and run a gel extraction.
- Ligation - Vary ratio of insert to backbone
Two insert samples
- Gel purification of most successful digest
- Unpurified sample of most successful digest
To backbone samples
- Gel purification of most successful digest
- Unpurified sample of most successful digest
For each combination creat samples
- 3ul backbone + 9 ul insert + ligase
- 1.5ul backbone + 15ul insert + Ligase
- Transform