Pooled Library Amplifications

By Hannah Dotson

Hannah Dotson May 30, 2024

If you’re depositing a pooled library with Addgene, you may be surprised to learn that we ask for an amplification protocol with your deposit. This is because repeated amplifications of pooled libraries can lead to issues such as recombination and loss of plasmid diversity, so Addgene strives to distribute aliquots of the original library whenever possible. In this blog, we’ll discuss how to develop an amplification protocol for your pooled library deposit.

When a requesting scientist receives a pooled library from Addgene, they’ll need to amplify the sample for further downstream applications. As individual libraries can vary significantly, it’s important that we have an amplification protocol developed for each individual pooled library; generalized amplification protocols tend not to work as well.

Defining your library

First, you’ll need to collect specific information about your library: the size of your library (How many unique plasmids does your library contain?), the variance of size of plasmids in your library (Are the inserts of different sizes?), and the desired coverage for the library (How important is it that every single plasmid in your library is present, and how will you ensure this?). You should also consider if there are any features of the library backbone that would make the library more difficult to work with. For example, a library in a lentiviral backbone might be more prone to recombination, while a library with a large backbone might be more difficult to transform.

Cartoon depicting factors to consider for a pooled library when creating an amplification protocol as described in the main text.

Figure 1: Factors to consider when creating a pooled library amplification protocol. Created with BioRender.com.

The most important factor to consider is your desired library coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled library protocols aim for somewhere between 30x and 200x coverage, meaning that you need 30 to 200 times as many colonies as individual plasmids in your library. As an example, if you want 100x coverage of your library, and your library contains 5,000 plasmids, your goal for your transformation would be to obtain 100 x 5,000 = 500,000 colonies.

Pro tip! Since you probably do not want to individually count 500,000 colonies, you’ll likely rely on serial dilutions to estimate transformation efficiency (Figure 2).

A schematic showing an example of a serial dilution. 10uL is used from an initial 1mL recovery and diluted into 990uL of LB. 100uL of this first dilution is plated for a 1:1000 dilution. 100uL from this first dilution are then diluted into another 900uL. 100uL of the second dilution is plated for a 1:10,000 dilution. This process is repeated twice more for 1:100,000 and 1:1,000,000 dilutions

Figure 2: Example of a serial dilution to estimate transformation efficiency. Figure used with permission from the Mark Dawson lab Re-transformation of the SPLINTR Libraries Protocol.

Selecting your strain for amplification

Since pooled libraries often contain thousands, if not millions, of plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible transformation efficiency. Using Stbl4 or other ultra-high efficiency cells ensures that plasmid diversity in the library is maintained. However, for smaller libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that of electroporation, but an advantage is that chemical transformations are generally cheaper and easier. One library that Addgene distributes with a chemical transformation amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library.

DNA and cell amounts

After deciding on a cell type, you will need to determine how much DNA and how many cells to use for your transformation(s). The amount of competent cells you need total and per transformation may vary depending on the particular type of cells you are using, as well as other factors, such as your lab’s normal transformation protocol, the size of the cuvettes your lab’s electroporator uses, etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the library, but generally needs to be determined experimentally. For larger libraries that require hundreds of nanograms of DNA, you will likely need to perform several individual transformations, such as splitting 400 ng of DNA in 100 μL of cells over four individual electroporations. The specifics of this can be based on the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically calculated based on the size of and weight of your library plasmids. As long as you achieve sufficient coverage from your transformation, any of these options work!

Selecting your media

Lastly, you need to decide between growing your library in liquid media or solid media (on plates). In general, amplification on plates can help to ensure that diversity of the library is maintained and reduce recombination during the amplification process. In liquid culture, all of the bacteria can grow and compete in the same container, making it easier for a smaller, recombined plasmid to overtake full-size plasmids (or for smaller plasmids to overtake larger plasmids, if the library varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony, and will not be in direct competition with other colonies containing smaller, recombined backbones that may replicate faster. For this reason, if your library has inserts of different sizes (such as a transcription factor overexpression library) or is prone to recombination, we recommend amplification on solid media. If your plasmid library is of a smaller size with a single size insert, liquid media can be faster and easier to work with. In either case, it can be useful to run a restriction digest of your amplified library on a gel to ensure that your conditions did not result in significant recombination. If you do see recombination, we recommend ensuring the cells used are meant for repetitive sequences (such as STBL4 or Endura Duo), growing your bacteria on solid media, reducing the incubation temperature (e.g., 30 °C), and limiting your growth time (e.g., 16-18 hours).

Finding reference protocols

There are many decisions to make when developing your amplification protocol! We recommend looking at protocols used for libraries that are similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can use as a reference.

Table 1: Examples of Different Pooled Libraries at Addgene.

Library Type	Name	Number of Plasmids	Variable Region Size	DNA per Amplification	Amplification Type	Link to Protocol
CRISPR Knockout	Human CRISPR Knockout Pooled Library (Brunello)	76,441	20 bp	400 ng	Solid	Library Amplification Protocol (PDF, 88.2 KB)
CRISPR Knockout	Human CRISPR Knockout Pooled Library (GeCKO v2)	123,411	20 bp	600–1200 ng	Solid	Joung et al. Nat Protoc. 2017
CRISPR Knockout	Human Epigenetic Knockout Library	20,051	20 bp	100 ng	Liquid	Library Amplification Protocol (DOCX, 15 KB)
CRISPR Activation	Human Genome-wide CRISPRa-v2 Libraries	104,540–209,080	20 bp	100 ng	Liquid	Liquid Culture Re-Amplification Protocol (PDF, 71 KB)
CRISPR Activation	Human Subpooled CRISPRa-v2 Libraries	12,285–20,145	20 bp	10–50 ng	Liquid	Liquid Culture Re-Amplification Protocol (PDF, 71 KB)
Barcode	Watermelon Library	5,000,000	30 bp	At least 200 ng	Liquid	Electroporation Protocol (DOCX, 15.8 KB)
Barcode	SPLINTR Pooled Libraries	200,000–1,200,000	60 bp	200 ng	Liquid	Re-transformation of the SPLINTR Libraries (PDF, 401 KB)
Screening	Zhang Lab - Multiplexed Overexpression of Regulatory Factors (MORF) Library	3,548	250 - 10,000 bp, plus a 24 bp barcode	250–1,000 ng	Solid	Joung et al. Nat Protoc. 2017 with modifications
Screening	pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) Pooled Library	80,962	1866 bp, plus a 20 bp barcode	400 ng	Solid	pCMV-Rep 78/68 SSM Library Amplification and Sequencing Protocol (DOCX, 32 KB)
Screening	Rinehart Lab Iterative Synthetically Phosphorylated Isomers (iSPI) Libraries	~11,000–110,139	144-188 bp	100 ng	Liquid	Library Amplification Protocol (DOCX, 17 KB)

For CRISPR libraries, Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab, and the Broad Institute’s general pDNA Library Amplification protocol are useful references.

Skim through our Pooled Library page to find other libraries that could be closer to your own pooled library. Be aware that some older libraries may not have amplification protocols attached.

Final step: validation

Remember, there is no “one-size-fits-all” protocol! Each library is different, and you should always experimentally test the protocol you design to ensure that it works for your library before depositing it with Addgene. If you have any specific questions about designing your library amplification protocol, feel free to contact us at deposit@addgene.org. Happy amplifying!