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

Today's gel

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.