Registry/Measurement kit/Preparation
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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
- 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
- <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
- 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 Endy:DNA Ligation. 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.
- TOP10 cells were transformed with prepped P1010.3k3 and no colonies were produced.