Endy:Library-based construction/Inverter library protocol: Difference between revisions
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#*[http://www.neb.com/nebecomm/products/productM0202.asp NEB] recommends a DNA concentration of 1-10 ug/ml (2-20 ng in a 20-ul reaction) for efficient circularization and subsequent transformation | #*[http://www.neb.com/nebecomm/products/productM0202.asp NEB] recommends a DNA concentration of 1-10 ug/ml (2-20 ng in a 20-ul reaction) for efficient circularization and subsequent transformation | ||
#Heat inactivate ligation reaction 10 min at 65 C unless you are using Quick Ligation Kit buffer, which contains PEG. If the ligation contains PEG, purify before using for transformation. | #Heat inactivate ligation reaction 10 min at 65 C unless you are using Quick Ligation Kit buffer, which contains PEG. If the ligation contains PEG, purify before using for transformation. | ||
#Dialyze the ligation product for | #Dialyze the ligation product for 30 mins | ||
==Transform into [[Electrocompetent cells|electrocompetent]] CW2553/pJat8== | ==Transform into [[Electrocompetent cells|electrocompetent]] CW2553/pJat8== |
Revision as of 11:14, 3 August 2006
We should be fleshing this out as we work through it a few times
Generate inverter library via mutagenic PCR
- 50 ul PCR reaction using Stratagene's GeneMorph® II Random Mutagenesis kit
- Amplifying VF2-VR
- Generally been using the "high" mutation frequency protocol, with ~20 ng initial target DNA
- PCR cleanup and elute with 30 ul
Digest the library and ligate into SP1.0
- Perform a 50 ul restriction digest using all of the PCR reaction
- Separate the insert out on a gel and perform gel extraction
- Elute the entire reaction into a single 30 ul volume (reuse on multiple columns if necessary)
- Perform a 20-ul ligation
- NEB recommends a DNA concentration of 1-10 ug/ml (2-20 ng in a 20-ul reaction) for efficient circularization and subsequent transformation
- Heat inactivate ligation reaction 10 min at 65 C unless you are using Quick Ligation Kit buffer, which contains PEG. If the ligation contains PEG, purify before using for transformation.
- Dialyze the ligation product for 30 mins
Transform into electrocompetent CW2553/pJat8
Controls
- (+): pUC18 (Amp resistance) (efficiencies)
- (-): cells only
Procedure
- Keep 2mm cuvettes on ice
- Thaw electrocompetent cells (40 ul per tube) on ice
- Add 1 ml of SOC to 12x75mm culture tube(s)
- Quickly spin down the thawed cells and add 3 ul (more, less?) of ligation product to the cells
- Keep on ice as much as possible
- Swirl with pipette tip to mix, pipetting is bad (heat, shear?)
- Add cell/DNA mix to the cuvettes in the center channel
- On the electroporation machine's menu, go to 4) pre-set protocols; 1) bacterial; 2) E.Coli-2mm, 2.5kV
- Should end up on a screen that lists the voltage and other parameters
- Should have a blinking 'P' in the corner of the screen
- Tap the cuvette on the table to settle cells to the bottom of the chamber, wipe the metal with a kim wipe and place in electroporation machine
- Press pulse button, wait, and check for a exponential decay graph with ~1800 volts applied and a time of ~4s
- If the screen goes back to the main menu, it worked
- If you hear a cracking noise/loud pop, sample arced, cells died, try again
- Quickly add the 1 ml of SOC from the culture tube and pipette up and down to get cells out of the chamber, then pipette everything back into the culture tube
- Grow for one hour without antibiotic
- Plate to determine library size and transformation efficiency
- plate a portion on selective media in order to determine the library size (50 ul = 1/20 of total library, could plate less depending on how big you think your library is)
- plate a portion on non-selective media in order to determine the number of surviving cells following electroporation (generally a serial dilution to 1/1e6 works fine)
- Move the rest of the culture into a higher volume (25 ml) of selective media
- Put the culture on a shaker in a baffled flask and allow the library to grow up overnight
Rest of procedure
- Store 5 glycerols of the libary from the overnight culture
- Spin down cells and switch to M9 media
- Measure OD to determine how many cells to add to your experimental cultures
- To be safe, take at least 10 times the library size (calculated from plates)
- Start experimental cultures with no induction and high induction
- Be sure to make a high enough dilution to allow for at least 9 doublings without going into stationary (OD600 < ~1)
- Grow overnight (rough doubling time is ~1.5-2 hrs)
- Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries
- Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like.
- Grow up the sub-libraries under the reverse condition (and the same condition)
- The reason for growing under the same conditions is to see how effective the sorting was (control, basically).
- Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions
- Grow up library and store 5 glycerols, as well as plate to get individual mutants
- Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library