User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/08

Karmella's BioBrick Cloning

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04/08/15 LCR development - test 5'-phos primers for Gal4DB-mCh-ATF2_MV10 assembly LCR - test 5'-phos primers Gal4DB-mCh (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1, R - mCh r1 ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1, R - ATF2_r1 MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1 Reagent Rxn1,2 Rxn3,4 Rxn5,6 Expected: 1,2. Gal4DB-mCh = 1170 3,4. ATF2 = 906 5,6. MV10 = 5191 10 μL/lane, 1% agarose; Ladder Template 0.2 2.0 2.0 10 uM fwd primer 1.0 1.0 1.0 10 uM rev primer 1.0 1.0 1.0 2x GoTaq green 12.5 12.5 12.5 dH2O 10.3 8.5 10.3   25.0 25.0 25.0 Program: GOTAQ35cyc 95°C, 3 min 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min] 72°C, 3 min 4°C ∞ Conclusions Gal4DB-mCh seems correct, except for spurious bands. Purify this product and re-run on gel ATF2 is veyr faint. Retry PCR with more cycles, lower temp. MV10 uppermost band might be correct. Try Phusion and gradient PCR.