Haynes Lab:Notebook/Jan/2015/04/06

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04/06/15 Assembly - DPRE into "BD004" Sequencing - BD006/MV2 Assembly *---Karmella 13:41, 6 April 2015 (EDT): BD004 appears to be missing the DPRE fragment. Gal4 sites and HPK are present (see alignment "BD004_P0001 (BD004_P0001.ab1)" on Benchling, https://benchling.com/s/0oSEQoHA/edit) Name it delBD004 BD004: insert DPRE(E/S)/1740 + vector delBD004(E/X)/7337 Use Roche 2x buffer and NEB T4 ligase Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/27 Calculations Vector - delBD004(E/X); 50 ng = 7.7 μL Insert - DPRE(E/S); (1740/7337)*2*50 = 23.7 ng = 1.4 μL Ligations DPRE + delBD004 delBD004 only (neg) Reagent 1 2 DNA insert 1.4 --- DNA vector 7.7 7.7 2x buffer 9.6 9.6 T4 ligase 0.5 0.5 dH2O --- 1.4   19.2 μL 19.2 μL --> RT/ ~30 min. Add 50 μL DH5-α turbo 5 min./ ice Plate on 100 ug/mL amp agar. Grow at 37 °C o/n RESULTS After ~7 hours of growth, saw tiny colonies only on ligation plate To save time, picked two colonies from ligation plate Make streaks on fresh agar Used tips to inoculate 5 mL cultures 4/07/15 Success! ~20 colonies in ligation plate, zero on negative ctrl. Growth on streak plate and in cultures Sequencing: BD006/MV2 Sample # Template Primer Benchling ref file Results (04/10/15) 1. BD006MV2_1 DD122B ChromatinSensor1_MV2 Good qual, t/C conflict at 605 2. BD006MV2_1 BGHR " Bad qual 3. BD006MV2_2 DD122B " Good qual, t/C conflict at 605 4. BD006MV2_2 BGHR " Good qual 5. BD006MV2_3 DD122B " Good qual, t/C conflict at 605 6. BD006MV2_3 BGHR " Good qual 7. BD006MV2_4 DD122B " Good qual, t/C conflict at 605 8. BD006MV2_4 BGHR " Good qual