CRISPR Library Prep Protocol: Difference between revisions
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>YanWu (Created page with ":Wet Lab #Using the selected bc_25mer primers, PCR the desired library using standard PCR protocol. #PCR purification. #Gel purification to remove other libraries (single ...") |
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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]]. | ||
#PCR purification. | #PCR purification. | ||
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#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. | #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]]. | #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 [[ | #Package vectors into lentivirus using [[Viral Packaging Protocol]] |
Revision as of 22:35, 5 May 2015
- Using the selected bc_25mer primers, PCR the desired library using standard PCR protocol.
- PCR purification.
- 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