TITLE: Pooled Library Amplification
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes pooled library amplification. To see the full abstract and additional resources, visit </div><div class = "text-block"><a href="https://www.addgene.org/protocols/pooled-library-amplification" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/pooled-library-amplification/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Day 1]
Add  DNA to each  aliquot of thawed Endura Duos on ice. Flick gently to mix
200 ng
50 µl
?. [Day 1]
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.
?. [Day 1]
Aliquot  DNA-Endura into pre-chilled cuvette.
25 µl
?. [Day 1]
Pulse.
Electroporation involves the use of high voltages, please use caution when activating pulse and follow all specifications described in the equipment manual.
?. [Day 1]
Immediately add  SOC to cuvette.
1 ml
?. [Day 1]
Remove all liquid from cuvette and add to  vented Falcon Tube containing  SOC.
14 ml
3 ml
?. [Day 1]
Repeat for each of the  aliquots of cell/DNA mixture used (8 in the described protocol) using a new vented falcon tube for every two transformations. Each of the four  vented falcon tubes should contain a total of  ( SOC +  transformed Endura from two separate transformations).
25 µl
14 ml
5 ml
3 ml
2 ml
*Pro-Tip* Do not pipette repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette.
?. [Day 1]
Shake four  Vented Falcon Tubes at -,  for .
14 ml
30 °C
37 °C
Centrifuge: 225 33
?. [Day 1]
After the 1 hour shaking period, pool and gently mix the four tubes.
?. [Day 1]
Perform sequential 1:100 dilutions of the cells (add  of the pool to  LB then perform a second and then third 1:100 dilution).  Plate  of each dilution onto a prewarmed Petri dish.
10 µl
990 µl
100 µl
?. [Day 1]
Incubate plates at  overnight.
30 °C
?. [Day 1]
Plate  of the transformed cells on each of the eight bioassay plates (two plates per tube). Distribute evenly with a sterile spreader until all liquid has been absorbed by the agar. This usually takes -.
2.5 ml
*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.
?. [Day 1]
Incubate plates upside down at  overnight.
30 °C
*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 1]
Place  sterile LB at .
100 ml
4 °C
?. [Day 2]
Before beginning, prechill at least four conical tubes on ice and ensure access to sterile scraper and cold LB.
*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.
?. [Day 2]
Count colonies on the most dilute Petri dish.
Total colony yield = count x 100 x 100 x 100 ÷ 0.1.This number should be at least 1000x greater than the number of perturbations in the 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).This number should be at least 1000x greater than the number of perturbations in the 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).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).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).
?. [Day 2]
After - of growth, use the spreader and cold LB to scrape bioassay plates and remove bacteria.
?. [Day 2]
Use one scraper for all plates.
?. [Day 2]
Use two  pipettes (one for dispensing cold LB and one for removing LB containing bacteria from the plate).
10 ml
*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.
?. [Day 2]
Add  cold LB to each plate for each scrape and use spreader to scrape plates.
10 ml
*Pro-Tip*Pushing motion is better than pulling motion and take care not to split or gouge agar during the scraping process.
?. [Day 2]
Add each scrape into a  conical tube on ice pooling the scrapings of two plates into each tube. Keep each tube on ice while scraping.
50 ml
?. [Day 2]
Repeat addition of  cold LB and scrape for each plate until agar is clear (all bacteria have been removed). This should produce  cold LB-bacteria per plate.
10 ml
25 ml
?. [Day 2]
Centrifuge tubes () to pellet bacteria.
Centrifuge: 4000 34, 15 min, 4 10
?. [Day 2]
Decant LB and weigh pellet. - The total weight of each pellet should be ~-.
1 g
2 g
*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.
?. [Day 2]
Purify plasmid DNA using the Qiagen HiSpeed Maxi Kit (one conical is its own 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.
?. [Day 2]
Quantify the individual Maxipreps by Nanodrop, Picogreen, or Qubit.
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.
?. [Day 2]
If all Maxipreps appear to contain sufficient DNA for use, pool samples, and continue with analysis.
“Sufficient” DNA will vary upon intended use. Typically yields range from several hundred micrograms to a milligram of total plasmid DNA.