Registry/Measurement kit/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

Components

• psb3K3
• <bbpart>E0240</bbpart>
• TOP10 Chemically competent cells

Preparation

psb3K3

Primers for Preparatory PCR

• Need to fill in here.

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:
  1. 95C for 5 min
  2. 95C for 30 sec
  3. 55C for 30 sec
  4. 68C for 3min 20sec
  5. Goto 2 35 times
  6. 68C for 10 min
  7. 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).

RBS Measurement Kit

Components

• psb3K3
• <bbpart>I13401</bbpart>
• TOP10 Chemically competent cells

Preparation

I13401

Miniprep

• Streak LB+Amp plate of I13401.1A2
• Innoculate 10ml of LB+Amp with a single colony from the plate, grow to saturation
• Miniprep the 10ml of culture (expected concentration?)
  • Load the entire sample into a single miniprep column
  • Elute with 50uL EB

Troubleshooting

• Described below are the tests we did to verify that the input parts to the measurement Kit are what we thought they are.