CRISPR Library Prep Protocol: Difference between revisions
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#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. | #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. | #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. | ||
#Using scaffold primers, PCR again with [[Scaffold primer PCR protocol]]. | #Using scaffold primers, PCR again with [[Scaffold primer PCR protocol]]. | ||
#PCR purification. | #PCR purification. |
Revision as of 03:47, 2 June 2015
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.
- Using scaffold primers, PCR again with Scaffold primer PCR protocol.
- PCR purification.
- Using Gibson Assembly Protocol ligate gRNAs into LGP 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. Keep one large culture (150 mL) for each library. 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. Maxi-prep rest of large culture to isolate DNA using Maxi-prep Protocol.
- Package vectors into lentivirus using Viral Packaging Protocol