Registry/Measurement kit/Preparation: Difference between revisions
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*Streak LB+Amp plate of I13401.1A2 | *Streak LB+Amp plate of I13401.1A2 | ||
*Innoculate 10ml of LB+Amp with a single colony from the plate, grow to saturation | *Innoculate 10ml of LB+Amp with a single colony from the plate, grow to saturation | ||
*Miniprep the 10ml of culture | *Miniprep the 10ml of culture | ||
**Load the entire sample into a single miniprep column | **Load the entire sample into a single miniprep column | ||
**Elute with 50uL EB | **Elute with 50uL EB | ||
*Measure the concentration of DNA in the sample (expected concentration?) | |||
====Digest==== | ====Digest==== | ||
*Load 1ug of DNA into a 50ml digest (X/P) | |||
====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?) | |||
=Troubleshooting= | =Troubleshooting= | ||
*Described below are the tests we did to verify that the input parts to the measurement Kit are what we thought they are. | *Described below are the tests we did to verify that the input parts to the measurement Kit are what we thought they are. | ||
Revision as of 13:01, 21 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
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:
- 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
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)
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?)
Troubleshooting
- Described below are the tests we did to verify that the input parts to the measurement Kit are what we thought they are.
