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]].
#PCR purification.
#PCR purification.
#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.
#PCR purification.
#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.
#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.
#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 [[Viral Packaging Protocol]]
#Package vectors into lentivirus using [[Viral Packaging Protocol]]

Revision as of 22:36, 5 May 2015

  1. Using the selected bc_25mer primers, PCR the desired library using standard PCR Protocol.
  2. PCR purification.
  3. Gel purification to remove other libraries (single stranded DNA) and any potential contaminants.
  4. Using scaffold primers, PCR again.
  5. PCR purification.
  6. 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.
  7. 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.
  8. 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.
  9. Package vectors into lentivirus using Viral Packaging Protocol