Haynes Lab:Notebook/Engineering PC-TFs/2012/02/16

Engineering PC-TFs

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Ligation/Assembly Weigh gel slices. Add three times amount of volume to each tube of gel (600 µL) Incubate at 55°C for 5-10 minutes. Centrifuge for a minute at 17,000 x g. Add 20 µL of wash buffer. Don't dump supernatent and centrifuge for one minute again. Place spin columns in 1.5 mL tubes. Measure concentration of each fragment: Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application Go to Nucleic Acid Quantification. Pipette 2 µL of each DNA sequence into Take 3 plate. Place plate in EPOCH plate reader. Results: Molarity of DNA Fragments 1. KAH01 96.477 ng/µL 2. KAH03 122.149 ng/µL 3. KAH04 80.182 ng/µL 4. KAH204 27.054 ng/µL 5. KAH205 47.105 ng/µL 6. KAH206 45.217 ng/µL 7. KAH225 66.047 ng/µL For all ligations, 25 ng of DNA vector = .3 µL There is a 2X molar ratio of insert:vector µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert) Assembly Scheme: Number beside vector and insert is number of base pairs 1. hPCD (186) + KAH204 (2328) 2. hPCD (186) + KAH205 (1251) 3. hPCD (186) + KAH206 (1551) 4. hPCD (186) + KAH225 (1251) 5. hPCD (186) only ' 1 2 3 4 5 DNA Insert 1.3 0.4 0.5 0.3 ----------- DNA Vector 0.3 0.3 0.3 0.3 0.3 T4 Ligase 1 1 1 1 1 Lign Buffer (2x) 5 5 5 5 5 dH2O 2.4 3.3 3.5 3.7 4 10 µL 10 µL 10 µL 10 µL 10 µL In a .5 mL tube, pipette the ligation buffer first. Then water, DNA insert, vector, and T4 ligase. Once completed, allow them to incubate for 10 minutes. Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes. Then follow transformation process: Transformation Process Warm 100 μg/mL Amp agar plates at 37 °C Thaw fresh tube of DH5α Turbo cells on ice Add 30 μL of DH5α Turbo cells to DNA. Incubate cells + DNA on ice for 5 min. Label pre-warmed plates Transfer cells + DNA onto agar Add 10 - 15 sterile glass beads, shake, discard beads Incubate plates at 37 °C overnight