6.3. NGS Sample Purification

To maximize sequencing of the targeted sgRNA or barcodes and minimize background sequences, we recommend purifying the amplified PCR products by preparative gel electrophoresis.

1. Based on quantitative analysis of PCR product yield for each of the samples, combine the PCR products from each sample at the same amount. For example, for samples with yields of correct PCR product of 20 ng/µl and 60 ng/µl, combine 6 µl and 2 µl of PCR reaction, respectively.

Purify and concentrate the PCR products from the combined sample using the QIAGEN QIAquick PCR Purification Kit, following the manufacturer’s protocol. The combined sample should be eluted in a volume of approximately 30 µl.

2. After purifying and concentrating, run each purified combined PCR product with 10X loading buffer on a 3.5% agarose-1XTAE gel with well sizes that accommodate at least 50 µl.

3. Using a transilluminator and scalpel, excise the narrow band that corresponds to the correct target sgRNA or barcode amplicon size.
   

CAUTION! Be sure to use UV safety glasses to protect your eyes when viewing and excising the DNA from the gel on the transilluminator.

4. After excision, purify the combined PCR product from the gel fragment using a QIAquick Gel Extraction kit following the manufacturer’s protocol. Elute the purified PCR product in 20 µl of elution buffer.

Note: Be sure to centrifuge QIAquick columns at maximum speed for at least 3 minutes before eluting DNA to avoid ethanol contamination in the purified PCR product.

5. Quantify extracted DNA in the combined sample by A260nm OD measurement using a NanoDrop spectrophotometer (or equivalent), and then adjust the concentrations in all samples to 10 nM. For example, if the amplicon size is 200bp, the 10nM concentration corresponds to 1.42 ng/µl based on A260 OD measurement. For an amplicon size of 750bp (dual-sgRNA product), a 10nM concentration corresponds to 5.3 ng/µl.

DNA samples can be stored at -20°C at this stage.

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