Haynes Lab:Notebook/Engineering PC-TFs/2015/01/12

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Summary Ligation Digest and Gel Verification Reagent Volume Expected: BL01/5 Ligation Colonies [XbaI/NruI] Forward insertion: 710, 7000 Reverse insertion: 3238, 4528 No insertion: 710, 4490 CMV/MV9 with [XbaI/NruI] 710, 4490 15 μL/lane; 1% agarose; Observed lengths: W2 BL05 LCR SV1 (small volume lcr reaction product: 4uL) W3 BL05 LCR SV2 W4 BL05 LCR LV1 (large volume lcr reaction product: 16uL) W5 BL05 LCR LV2 W6 BL01 LCR SV1 W7 BL01 LCR SV2 W8 Empty CMV/MV9 W9 BL01 LCR LV1 W10 BL01 LCR LV2 W11 LCR Negative Control Colony W12 BL05 Traditional Ligation SV1 W13 BL05 Traditional Ligation SV2 W14 BL05 Traditional Ligation LV1 Ladder DNA 4.0 μL 10X buffer 2.0 μL XbaI 1.0 μL NruI 1.0 μL dH2O 12 μL Total 20 μL --> 37°C/ 30 min. Well 9 (BL01 LCR) looks promising! Will sequence after remaining samples are analyzed. Using DD123 & DD122 primers, a forward insertion will result in a band of length 3371 running the PCR reaction.