Registry/Measurement kit/Preparation: Difference between revisions
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Description of the measurement kit components and methods of preparation. | Description of the measurement kit components and methods of preparation. See also [[Registry/Measurement kit/Instructions]] for a description of how to use the kit to test a promoter or RBS. | ||
=Promoter Measurement Kit= | =Promoter Measurement Kit= | ||
Revision as of 08:55, 14 January 2008
Description of the measurement kit components and methods of preparation. See also Registry/Measurement kit/Instructions for a description of how to use the kit to test a promoter or RBS.
Promoter Measurement Kit
Components
- psb3K3
- <bbpart>E0240</bbpart>
- TOP10 Chemically competent cells
Preparation
psb3K3
Preparatory PCR
Result of a successful preparatory PCR; Ladder is 1ug of 2log
- The psb3K3 "backbone" plasmid is produced by PCR from a prepped psb3K3 template.
- A preparative PCR mix containing:
- 190 of PCR Supermix HIFI from Invitrogen
- 5uL of 3K3SuffixFWD primer
- 5uL of 3K3PrefixREV primer
- 0.5uL of template at ~40ng/uL
- Run PCR with the following conditions:
- 95C for 5 min
- 95C for 30 sec
- 55C for 30 sec
- 68C for 3min 20sec
- Goto 2 35 times
- 68C for 10 min
- 4C for eva
- Notes: I haven't really optimized the PCR conditions, but I find that using the Supermix is much more reliable than using the random endy lab stocks, fwiw.
PCR Cleanup
- PCR Cleanup on a 200uL preparative PCR mix should yield about 50uL of cleaned up DNA at a concentration of ~330ng/uL (16.5ug).
Restriction Digest
RBS Measurement Kit
Components
- psb3K3
- <bbpart>I13401</bbpart>
- TOP10 Chemically competent cells
