Here is a list of components that are needed for this project:
- Anchor DNA:
- pRL574 – provided to us by Robert Landick
- pALS – purchased through DNA 2.0
- primers for both pRL574 and pALS
- F834-dig: 5′ – TTTTCCCAGTCACGACGTTG – 3′
- R2008-bio: 5′ – CACGTAAGGTTTCAGAGATATATGGG – 3′
- F50 (can order with or without Biotin): 5′ – TGTGTCGCCCTTAGGTACGAACT – 3′
- R4000 (can order with or without dig): 5′ – TTCGCTCCAAGCTGGGCTGTGTG – 3′
- Adapter DNA:
- Top Adapter: 5′ – GCTGTCTGAATTCTAATGTAGTATAGTAATCCGCTCATCG – 3′
- make sure the 5′ end is phosphorylated otherwise the ligation may fail
- Bottom 1a: 5′ – GAGCGGATXACTATACTACATTAGAATTCAGAC -3′
- X = dT-biotin
- Alternate versions of Bottom
- 5′ – TXTXTXAGAGCGGATTACTATACTACATTAGAATTCAGAC -3′
- X = dT-biotin
- 5′ – biotin – GAGCGGATTACTATACTACATTAGAATTCAGAC – 3′
- 5′ – TXTXTXAGAGCGGATTACTATACTACATTAGAATTCAGAC -3′
- Top Adapter: 5′ – GCTGTCTGAATTCTAATGTAGTATAGTAATCCGCTCATCG – 3′
- Molecular Biology Components
- PCR Supplies:
- Taq/OneTaq – I had amazing success with OneTaq so I may get this
- dNTPs
- MgCl2
- PCR buffer
- Digestion components
- BstXI
- SapI
- EarI
- Ligation
- T4 DNA Ligase
- Gel Components
- EtBr
- SybrSafe
- PCR Supplies: