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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]]
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