- Q5 Master Mix (New England Biolabs)
- Forward and Reverse primers (10 uM) (See below)
- synthetic template DNA (See below)
- Sterile microcentrifuge tubes and PCR tubes
The sequence of the synthetic template DNA used:
CCAACTTAAT CGCCTTGCGC CGCCAGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC GTTGCGCAG CCTGAATGGC GAATGGCGCT TTGCCTGGTA TCCTGCACCA GGAGCGCTGC CGGAAAGCTG GCTGGAGTGC ATCTTCCTG AGGCCGATAC TGTCGTCGTC CCCTCAAACT GGCAGATGCA CGGTTACGAT GCGCCCATCT ACACCAACGT GACCTATCCC ATTACGGTCA ATCCGCCGTT TGTTCCCACC TAGAATCCGA CGGGTTGTTA CTCGCTCACA TTTAATGTTG ATGAAAGCTG GCTACAGGAA GGCCAGACGC GAATTATTTT TGATGGCGTT CCTATTGGTT AAAAAATGGATATCGATCCACGGATCCAGG TGCGAATTTT AACAAAATAT TTACGGTTAC AATTTGGGTA TTTGCTTATA CAATCTTCCT GTTTTTGGGG CTTTTCTGAT TATCAACCGG GGTACATATG AATTCCATGC TAGTTTTACG ATTACCGTTC TTCGTTTCTC TTGTTTGCTC CAGACTCTCA GGCAATGACC TGATAGCCTT TGTAGATCTC TCAAAAATAG GGATATCGATCCACGGATCC TGGTCAGCTA GAACGGTTGA ATATCATATT GATGGTGATT TGACTGTCTC CGGCCTTTCT CACCCTTTTG AATCTTTACC TACACATTAC TCAGGCATTG CAGGGAAAAT ATATGAGGGT TCTAAAAATT TTTATCCTTG CGTTGAAATA AAGGCTTCTC CCGCAAAAGT ATTACAGGGT CATAATGTTT TTGGTACAAC CGATTTAGCT TTATGCTCTG AGGCTTTATT GCTTAATTTT GCTAATTCTT TGCCTTGCCT GTATGATTTA TTGGATGTTA ATGCTACTAC TATTAGTAGA ATTGATGCCA CCTTTTCAGC TCGCGCCCCA AATGAAAATA TAGCTAAACA GGTTATTGAG CGAAATGTAT CTAATGGTCA
The forward primer sequence: 5’-CGCGTTGGCCGATTC-3’
The reverse primer sequence: 5’-AGGGTGAGAAAGGCC-3’.
- PCR product from Step 5
- Qiagen PCR clean up kit (with spin columns and all reagents)
- Microcentrifuge tube for storage of purified DNA
- Purified DNA from Step 7
- Water or EB buffer from PCR cleanup kit
- Nanodrop spectrophotometer
- Sterile nuclease-free microfuge tubes
- Nuclease-free water
- EnGen 2X sgRNA Reaction Mix, S. pyogenes
- Target-specific DNA oligo (1 µM) (see below)
- Create by adding 1 µL oligo into 99 µL RNase free water
- DTT (0.1 M)
- EnGen sgRNA Enzyme Mix
The sequence of the target specific DNA oligo:
TTCTAATACGACTCACTATAGGATATCGATCCACGGATCCGTTTTAGAGCTAGA
- RNA product from Synthesis protocol above
- Monarch RNA Cleanup Kit (50 µg)
- Microcentrifuge tube for storage of purified RNA
- Sterile nuclease-free microcentrifuge tubes
- Dilute product from sgRNA synthesis with nuclease-free water
- 1 µM dCas9, S. pyogenes