CRISPR Library Prep Protocol
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Note: Every time you pipette from a library, always pipette multiple times. For example, if you are pipetting 5 uL of library, do 5 x 1 uL liquid transfers instead of 1 5 uL liquid transfer. This helps ensure a more uniform library coverage and reduces the probability of a gRNA dropout.
- Using the selected bc_25mer primers, PCR the desired library using the Barcode primer PCR protocol. Use at least 2 PCR reactions per library. For large libraries (more than 1000 gRNAs), use up to 4 PCR reactions.
- Gel purification to remove other libraries (single stranded DNA) and any potential contaminants. Use a 2% agarose gel. Be careful not to run the gel too long since it's very easy for the small PCR product to run off of the gel.
- Dilute product from step 2 to < 1 ng/uL. Using the barcoding scaffold primers, run 4 cycles of PCR.
- Run an Exo treatment on the product & then 1.8:1 beads purification
- Using the scaffold PCR only primers, amplify the product until you get a clear band
- Gel purification
- Using Gibson Assembly Protocol ligate gRNAs into desired vector, run gel to check ligation worked. Note: include control LGP assembly with no insert to check for self-ligation.
- Transform into chemically competent E.Coli cells using Transformation Protocol. Plate each library onto large (15 cm plates) to reduce amplification bias. Also plate 4 plates per library and 2 plates total as a control.
- Check plates and count colonies. Need at least 10x cells for proper coverage. (note: be more specific about math). Keep 16 1.5 mL tubes of large culture for frozen stock. Scrape the colonies off of the large culture plates using LB and then follow standard Maxi-prep Protocol.
- Package vectors into lentivirus using Viral Packaging Protocol