CRISPR Library Prep Protocol: Difference between revisions
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#Using the selected bc_25mer primers, PCR the desired library using standard [[PCR Protocol]]. | #Using the selected bc_25mer primers, PCR the desired library using standard [[PCR Protocol]]. | ||
#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. | ||
#Using scaffold primers, PCR again. | #Using scaffold primers, PCR again. |
Revision as of 22:26, 6 May 2015
- Using the selected bc_25mer primers, PCR the desired library using standard PCR Protocol.
- Gel purification to remove other libraries (single stranded DNA) and any potential contaminants.
- Using scaffold primers, PCR again.
- 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