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
 * E0240
 * TOP10 Chemically competent cells
 * 3k3SuffFWD: TAC TAG TAG CGG CCG CTG CAG
 * 3k3PreREV: CTC TAG AAG CGG CCG CGA ATT C

Primers for Preparatory PCR

 * 3k3SuffFWD: TAC TAG TAG CGG CCG CTG CAG
 * 3k3PreREV: CTC TAG AAG CGG CCG CGA ATT C

Preparatory PCR

 * [[Image:Psb3k3 Pcr prep.JPG|thumb|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 48uL of cleaned up DNA at a concentration of ~330-350ng/uL (16ug).

Restriction Digest

 * Load 1ug of DNA into a 50ml digest (X/P)
 * I use this protocol (except use buffer NEB3)

=RBS Measurement Kit=

Components

 * psb3K3
 * I13401
 * TOP10 Chemically competent cells
 * 3k3SuffFWD: TAC TAG TAG CGG CCG CTG CAG
 * 3k3PreREV: CTC TAG AAG CGG CCG CGA ATT C

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
 * Load the entire sample into a single miniprep column
 * Elute with 50uL EB
 * Measure the concentration of DNA in the sample (expected concentration?)

Digest

 * Load 1ug of DNA into a 50ml digest (X/P)
 * I use this protocol (except use buffer NEB3)

PCR Cleanup

 * Expected conecntation
 * On average we see ~65% yield between what went into the digest and what comes out of this cleanup step.
 * So expected concentration will be ~22ng/uL in 30uL elution.
 * I load 3uL into the ligation, so if you need to do more than 9 ligations then make up multiple digests (or include more DNA in the digest -- up to how much?)

Ligation

 * Most recently I tried 10ng of each of the inputs (I13401, psb3K3, and P.RBS) in a 20uL ligation reaction with concentrations as specified in the Endy lab protocol. This hasn't been optimized, but I ran some controls (number of colonies after ='s):
 * 3k3 PCR-product (non-ligated) = 3
 * 3k3 PCR-product digested E/P (non-ligated) = 6
 * 3k3 PCR-product digested E/P (ligated) = 1
 * 3k3 + I13401 = 26
 * 3k3 + I13401 + b0031 = 1
 * 3k3 + I13401 + b0033 = 56
 * 3k3 + I13401 + b0034 = 19
 * Not sure exactly what to make of this, clearly some more optimization is needed. One point to note is that I only directly measured the concentration of one of the P.RBS annealed primer preps (though they all should be at the same concentrations, should dbl check the others).

=Troubleshooting=
 * Described below are the tests we did to verify that the input parts to the measurement Kit are what we thought they are.
 * TOP10 cells were transformed with prepped P1010.3k3 and no colonies were produced.
 * This is to demonstrate that any P1010.3k3 used as template for the PCR reaction will not appear as background false-positive colonies when doing the final ligation/transformation.