Haynes Lab:Notebook/Engineering PC-TFs/2015/02/26

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Summary Restriction Digest/Gel Verification, Concentration Reading Digest and Gel Verification Reagent Volume Expected: BL01/5 Ligation Colonies [XbaI/NruI] Forward insertion: 3659, 4064 Reverse insertion: 1538, 6185 No insertion: 1538, 4064 CMV/MV9 with [XbaI/NruI] 1538, 4064 Uncut CMV/MV9: 5197 Uncut CMV/BL05/MV9: 7723 15 μL/lane; 1% agarose; Observed lengths: W1: Ladder W2: Ryan's stuff W3: BL05 LCR W4: BL05 LCR W5: BL05 LCR W6: BL05 LCR W7: BL05 LCR W8: BL05 LCR W9: BL05 LCR W10: BL05 LCR W11: BL05 LCR W12: BL05 LCR W13: BL05 LCR W14: BL05 LCR Ladder DNA 4.0 μL 10X buffer 1.5 μL XbaI 1.0 μL BsmbI 0.5 μL dH2O 8 μL Total 15 μL --> 37°C/ 20 min. Looks as though one enzyme perhaps didn't cut or was not added. Empty wells probably a result of bad amp. DNA in well 13 will be re-examined as it seems to be bigger than the others. Possible insertion.