Registry/Measurement kit/Notebook/2007-6-15

Plan for today

 * 1) Digest constructs
 * 2) Ligate components
 * 3) 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
See proposed solution at Registry/Measurement_kit/Notebook/Design
 * 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)
 * 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:
 * 1) R0040
 * 2) B0032
 * 3) I13401
 * 4) 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 B: GFP RBS tester C: RFP RBS tester
 * B0032 + J04650
 * R0040 + I13401
 * R0040 + J0465

Transformation of devices

 * Attempting to transform the ligated devices.
 * Strain - MG1655