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

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  • Restriction Digest/Gel Verification, Concentration Reading

Digest and Gel Verification

Reagent Volume

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

Today's gel

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

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.