Pooled Library Amplification
This protocol allows the amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used for screening, barcoding, or other high throughput multiplexed experiments. These pooled plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's genome, for example.
Amplification is usually necessary to produce sufficient quantities of library for experimental applications. Repeated amplifications should be avoided as best as possible due to the inherent possibility of altering the composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the representation of individual plasmids in the pooled library during amplification. This protocol is designed to be as general as possible but note that individual libraries may require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled library from Addgene, these protocols can be found on the Pooled Library page for the specific library.
This protocol assumes familiarity with standard bacterial transformation and basic knowledge of the library being amplified. Please see our protocol on Bacterial Transformation. The generalized pooled library amplification protocol is expected to obtain satisfactory results for libraries up to 200,000 individual plasmids and can be scaled according to the actual number.
Required Quality Control (QC)
Pooled libraries can be challenging and expensive to ensure adequate quality, but this upfront cost will save headaches and expense later.
At a minimum we recommend the use of a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme for digest that will cut the shared plasmid backbone a single time and visualize that digestion on an agarose gel (see restriction digest protocol here). Lentiviral plasmids can recombine between their LTRs resulting in a smaller plasmid containing only the elements required for bacterial propagation (origin of replication and antibiotic selection). This recombination, at a low rate, is not typically a problem as these sequences are not efficiently packaged into lentiviral particles. See below for options if the recombinant band makes up a significant proportion of the DNA pool.
Last Update: February 27, 2020
- Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours)
- Transformation should be performed at the end of the day to ensure that growth times are limited.
- Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours)
- Cells should be harvested first thing in the morning to ensure that outgrowth and competition are limited.
- Table top centrifuge
- BioRad Electroporator (MicroPulser TM , Bio-Rad 1652100)
- 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1)
- Alternatives include Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone DNA.
- The use of electrocompetent cells is essential to ensure high efficiency uptake of plasmid library DNA.
- This quantity of cells is sufficient for libraries up to 200,000 individual plasmids. For larger libraries scale the number of cells and DNA accordingly.
- 800 ng pooled library DNA (100 ng plasmid DNA per 25 µL electrocompetent cells)
- Less library DNA can be used but this dramatically increases the chances of individual plasmids being lost from the pool and/or increasing the skewness of the pool.
- 8 electroporation cuvettes (BioRad, Micropulser, 0.1 cm )
- 20 mL SOC recovery media (Lucigen, 80026-1)
- 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular Devices, X602)
- 14 mL Vented Falcon Tubes (BD Biosciences, 352059)
- 3X LB Agar + Antibiotic 65 mm Petri dish (VWR, 11019-552)
4 MaxiPreps (Qiagen HiSpeed Max, Catalog #12663)
- Tips (1000 µL, 200 µL, 10 µL)
- Bacti Cell Spreaders (VWR, 60828-680)
- 5 mL and 10 mL Serological pipettes
- Ice slurry (Ice bucket with ice and water to create slurry)
- 100 mL LB
- At Addgene we use premixed LB (VWR 101414-072) for convenience but any brand or house- made LB that supports normal growth is expected to work.
- 50 mL Falcon Conical tubes (Fisher, 14-432-22)
*Pro-Tip* Use of extreme high efficiency (1x1010 cfu/µg) commercially prepared electrocompetent cells is strongly recommended.
*Pro-Tip* Pour these plates at least one day in advance to allow adequate time to fully gel and to dry slightly. We routinely use 350 mL of LB Agar per bioassay plate.
- Prepare, sterilize, and pour all LB Agar + Antibiotic plates.
- Prewarm 12 mL recovery media at 37 °C (for at least 15 minutes).
- Prewarm 3X LB Agar + Antibiotic plates at 37 °C.
- Prewarm 8X LB Agar + Antibiotic Bioassay plates.
- Prechill Micropulser cuvettes on ice.
- Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely thawed.
- Chill a box of 200 µL micropipette tips in a -20 °C freezer.
- Aliquot 3 mL SOC into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation recovery of cells.
- Ensure access to autoclaved, sterile reagents for all steps.
- Add 200 ng DNA to each 50 µL aliquot of thawed Endura Duos on ice. Flick gently to mix.
- Electroporate cells (one at a time for a total of eight electroporations):
- Electroporator Conditions: Bio-Rad Micropulser Ec1 0.1 cm cuvette, 1.8 kV, 1 pulse.
- *Caution* Electroporation involves the use of high voltages, please use caution when activating pulse and follow all specifications described in the equipment manual.
*Pro-Tip* Do not pipette repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette.
- *Critical* Be careful not to rip or shred the agar. Do so by gentle spreading. Some spreaders have a sharp edge that can scrape plates more abrasively at a certain angle.
- *Critical* Ensure at this stage that no unabsorbed media drips onto the lid. Let plates remain agar side up until dried before overnight incubation if needed.
Day 2 (morning)
- Before beginning, prechill at least four conical tubes on ice and ensure access to sterile scraper and cold LB.
- Count colonies on the most dilute Petri dish.
*Pro-Tip* Prepare one to two more conical tubes on ice in case you need to spread out the harvested cells further than four Maxipreps worth.
- Total colony yield = count x 100 x 100 x 100 ÷ 0.1
- This number should be at least 1000X greater than number of perturbations in library.
- Example: At least 10 colonies on the most diluted plate for 1000x coverage of a library of 100,000 plasmids.
- Colonies may appear small and require extra incubation time in order to be enumerated accurately.
- Frequently the number of colonies can be too great to count. Ideally, dilutions would have been sufficient to enumerate single colonies but as long as one can ensure more than the required colonies are present. If colonies are not present on the most dilute plate, count the second plate (ie. the 1:10,000 dilution).
*Pro-Tip* Scraped bacteria in LB can clog the pipette either mix gently up and down avoiding introducing bubbles or pour off plates into conical tubes as needed.
*Pro-Tip* Pushing motion is better than pulling motion Take care not to split or gouge agar during the scraping process.
*Pro-Tip* Make sure to weigh the empty tube beforehand! If you’ve already gone too far, weigh an identical empty tube. It will be close enough for the purpose of a Maxiprep.
- *Critical* Do not freeze pellets for later purification. Immediately purify them! Commercial Maxipreps rely on incremental, ordered cell lysis. E. coli cells are subject to lysis by freeze-thaw if not suspended in cryoprotectant like Glycerol or DMSO solutions.
- Each different DNA measurement type can report slightly different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than Nanodrop, although most protocols have been designed with Nanodrop as the quantification method.
- “Sufficient” DNA will vary upon intended use. Typically yields range from several hundred micrograms to a milligram of total plasmid DNA.