Registry/Measurement kit/Notebook/2007-6-15
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Plan for today
- Digest constructs
- Ligate components
- Transform
Double Digest
- See protocol
- Used buffer 3 as suggested by NEB for the XbaI/PstI digests
- Used 2uL stock R0040 instead of prepped
- 22.12 uL of B0032
- 8.84 uL of I13401
- 4.68 uL of J04650
- Incubated at 37 for 5 hrs
Design discussion
- Inserting a standard BioBrick site (...EcoRI-XbaI-[ccdB]-SpeI-PstI...) for the placement of an RBS would leave too big a gap between the RBS and translation initiator (http://parts.mit.edu/registry/index.php/Assembly:RBS-CDS_issues)
See proposed solution at Registry/Measurement_kit/Notebook/Design
- It was suggested that we use the LacZ reporter gene instead of an FP, but all that appears to be in the registry is an N-terminus fragment of the LacZ gene. In the near future, we plan to standardize and enter the LacZ gene into the registry.
PCR purification
- buffer transfer of cut DNA
- protocol is in booklet with the kit
- note: labeling system for digests:
- R0040
- B0032
- I13401
- J04650
Ligation
Endy:DNA_ligation_using_T4_DNA_ligase
- Used modified version of above protocol. Will post on OWW soon.
Labels: A: RFP promoter tester
- B0032 + J04650
B: GFP RBS tester
- R0040 + I13401
C: RFP RBS tester
- R0040 + J0465
Transformation of devices
- Attempting to transform the ligated devices.
- Strain - MG1655
