CRISPR Library Amplification

Day 1

1. Add 200 ng DNA to each 50 µL aliquot of thawed Endura Duos on ice. Flick gently to mix.
2. 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.
3. Aliquot 25 µL DNA-Endura into pre-chilled cuvette.
4. Pulse.
• *Caution* Electroporation involves the use of high voltages, please use caution when activating pulse and follow all specifications described in the equipment manual.
5. Immediately add 1 mL SOC to cuvette.
6. Remove all liquid from cuvette and add to 14 mL vented Falcon Tube containing 3 mL SOC.
7. Repeat for each of the 25 µl 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 14 mL Vented Falcon Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations).

*Pro-Tip* Do not pipette repeatedly or mix when removing SOC containing transformed DNA-Endura from cuvette.

8. Shake four 14 mL Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour.
9. After the 1 hour shaking period, pool and gently mix the four tubes.
10. Perform sequential 1:100 dilutions of the cells (add 10 µL of the pool to 990 µL LB then perform a second and then third 1:100 dilution). Plate 100 µL of each dilution onto a prewarmed Petri dish.
11. Incubate plates at 30 ℃ overnight.
12. Plate 2.5 mL 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 1-2 minutes.
• *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.
13. Incubate plates upside down at 30 ℃ overnight.
• *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.
14. Place 100 mL sterile LB at 4 ℃.

Day 2 (morning)

15. 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.

16. 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 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).
17. After 12-18 hours of growth, use spreader and cold LB to scrape bioassay plates and remove bacteria.
18. Use one scraper for all plates.
19. Use two 10 mL pipettes (one for dispensing cold LB and one for removing LB containing bacteria from plate).

*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.

20. Add 10 mL cold LB to each plate for each scrape and use spreader to scrape plates.

*Pro-Tip* Pushing motion is better than pulling motion Take care not to split or gouge agar during the scraping process.

21. Add each scrape into a 50 mL conical tube on ice pooling the scrapings of two plates into each tube. Keep each tube on ice while scraping.
22. Repeat addition of 10 mL cold LB and scrape for each plate until agar is clear (all bacteria have been removed). This should produce 25 mL cold LB-bacteria per plate.
23. Centrifuge tubes (4000 G, 4 ℃, 15 minutes) to pellet bacteria.
24. Decant LB and weigh pellet. The total weight of each pellet should be ~1-2 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.

25. 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.
26. 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.
27. 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.

What do I do if my transformation efficiency is not high enough?

Repeat the experiment with more cells, more DNA, gentler practices, and colder reagents (not including the recovery media!). Do not proceed with Maxipreps or NGS until adequate transformation efficiency is obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide adequate transformation efficiency! Ensure that no arcing is taking place during electroporation. Arcing would manifest as a loud pop often accompanied by some light in the cuvette during the electroporation step.

I got a heavy recombined band by digest.

Recombination is thought to be in part related to selective pressure. These recombined plasmids are much smaller and are a smaller nutritional burden on the bacteria. Bacteria that contain a recombinant plasmid likely grow faster in the closed system of a crowded plate or culture. Try restricting growth to the bare minimum by growing at 30 ℃, limiting growth time further, or potentially spreading less bacteria on a given plate (example: by increasing the total number of bioassay plates used). Scaling up the experiment using a Gigaprep can be used to obtain sufficient intact pooled library even in the face of a recombined fraction but care must be taken to perform adequate NGS based QC to ensure no change in representation compared to the pre-amplified stock. A last option is to perform a gel extraction of either the original sample or the amplified sample, followed by reamplification of the DNA, but please note that NGS should be performed to ensure representation is maintained.

Maxipreps - Less is more:

Do not overload the Maxipreps as yield can dramatically plateau and sometimes fall off entirely if the column or reagents are significantly overloaded. If you find that your pellet outstrips the capacity listed in the Maxiprep protocol, scale the reagent volume and column number as needed. The use of Mega or Gigapreps is acceptable when scaling up or reducing the number of tandem purifications. Consult the manufacturers handbooks for appropriate volumes and numbers of tandem purifications.