February, 2023
Genomic DNA Preparation by Macherey-Nagel Tissue Spin Columns
Using this kit according to the manufacturer’s protocol is the standard way for preparing genomic DNA at OTTC when it is intended for ddPCR (i.e. copy-number quantification).
It will also work for conventional PCR genotyping. Typically, 10 to 90 ng of genomic DNA is used in a 11uL PCR reaction.
General PCR reaction setup
5.0 uL 2x OneTaq Hot Start 2x Master Mix with GC Buffer (New England Biolabs)
5.0 uL 2x specific oligos (Forward & Reverse primer, 1 micromolar each, in water)
1.0 uL genomic DNA
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Total: 11.0 uL PCR reaction
Oprm1 Gen For | CATATTTATGATGGCCCTCATTCTGAGC | 5' junction |
---|---|---|
iCre R408 | CCCAGCATCCACATTCTCCTT | 5' junction |
iCre F102 | CCTGATGGACATGTTCAGGG | 3' junction |
Oprm1 Gen Rev | CTGTACATGAGCTGGATTCAGGAGTG | 3' junction |
Oprm1 Gen For | CATATTTATGATGGCCCTCATTCTGAGC | WT/Tg |
Oprm1 Gen Rev | CTGTACATGAGCTGGATTCAGGAGTG | WT/Tg |
5’ junction
These oligos (Oprm1 Gen For and iCre R408) produce a 649 basepair (bp) amplicon in samples that carry the transgene (Tg).
3’ junction
These oligos (iCre F102 and Oprm1 Gen Rev) produce a 1187 bp amplicon in samples that carry the transgene (Tg).
WT/Tg
These oligos (Oprm1 Gen For and Oprm1 Gen Rev) produce a 416 bp amplicon in wildtype (WT) samples, and a 1529 bp amplicon in samples that carry the transgene (Tg). NOTE: While this assay works to detect both the WT allele and the Tg allele, amplification of the WT amplicon outcompetes the Tg amplicon in the heterozygous samples.
Reagents/Kits used
Macherey-Nagel – 740952.10 – NucleoSpin Tissue
New England Biolabs – M0485S – OneTaq Hot Start 2x Master Mix with GC Buffer
Sequences
>OPRM1_rat_WT_amplicon
catatttatgatggccctcattctgagctcccaacaaaagcacaagacaatgttcagtacagttctcatacccagaagacaatcatcaatgtgctctctaatgagaccccagaactcactatcttcactctttctcttctttcagctagaaaatctggaggcagaaactgctccattgccctaactgggtctcacaccatccagaccctcgctaagcttagaggccgccatctacgtggaatcaggttgctgtcagggtgtgtgggaggctctggtttcctgagaaaccatctgatcctgcattcaaagtcattcctctctggctacttcactctgcacatgagagatgctcagactgtatcaagtactcagaaagaagagactaccggacactcctgaatccagctcatgtacag
>OPRM1-T2A-ICRE_rat_amplicon_1529nuc
catatttatgatggccctcattctgagctcccaacaaaagcacaagacaatgttcagtacagttctcatacccagaagacaatcatcaatgtgctctctaatgagaccccagaactcactatcttcactctttctcttctttcagctagAAAATCTGGAGGCAGAAACTGCTCCATTGCCCGGTTCTGGCgagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctGTGCCCAAGAAGAAGAGGAAAGTCTCCAACCTGCTGACTGTGCACCAAAACCTGCCTGCCCTCCCTGTGGATGCCACCTCTGATGAAGTCAGGAAGAACCTGATGGACATGTTCAGGGACAGGCAGGCCTTCTCTGAACACACCTGGAAGATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCCTGGTGCAAGCTGAACAACAGGAAATGGTTCCCTGCTGAACCTGAGGATGTGAGGGACTACCTCCTGTACCTGCAAGCCAGAGGCCTGGCTGTGAAGACCATCCAACAGCACCTGGGCCAGCTCAACATGCTGCACAGGAGATCTGGCCTGCCTCGCCCTTCTGACTCCAATGCTGTGTCCCTGGTGATGAGGAGAATCAGAAAGGAGAATGTGGATGCTGGGGAGAGAGCCAAGCAGGCCCTGGCCTTTGAACGCACTGACTTTGACCAAGTCAGATCCCTGATGGAGAACTCTGACAGATGCCAGGACATCAGGAACCTGGCCTTCCTGGGCATTGCCTACAACACCCTGCTGCGCATTGCCGAAATTGCCAGAATCAGAGTGAAGGACATCTCCCGCACCGATGGTGGGAGAATGCTGATCCACATTGGCAGGACCAAGACCCTGGTGTCCACAGCTGGTGTGGAGAAGGCCCTGTCCCTGGGGGTTACCAAGCTGGTGGAGAGATGGATCTCTGTGTCTGGTGTGGCTGATGACCCCAACAACTACCTGTTCTGCCGGGTCAGAAAGAATGGTGTGGCTGCCCCTTCTGCCACCTCCCAACTGTCCACCCGGGCCCTGGAAGGGATCTTTGAGGCCACCCACCGCCTGATCTATGGTGCCAAGGATGACTCTGGGCAGAGATACCTGGCCTGGTCTGGCCACTCTGCCAGAGTGGGTGCTGCCAGGGACATGGCCAGGGCTGGTGTGTCCATCCCTGAAATCATGCAGGCTGGTGGCTGGACCAATGTGAACATTGTGATGAACTACATCAGAAACCTGGACTCTGAGACTGGGGCCATGGTGAGGCTGCTCGAGGATGGGGACTAACTGGGTCTCACACCATCCAGACCCTCGCTAAgcttagaggccgccatctacgtggaatcaggttgctgtcagggtgtgtgggaggctctggtttcctgagaaaccatctgatcctgcattcaaagtcattcctctctggctacttcactctgcacatgagagatgctcagactgtatcaagtactcagaaagaagagactaccggacactcctgaatccagctcatgtacag
This protocol was updated on February 2023. Any questions regarding protocol, contact nidatransgenicprojects@mail.nih.gov.