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

• 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:

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.