Haynes Lab:Notebook/Engineering PC-TFs/2015/01/19

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Summary

Ligation 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

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 NEG
W6 BL01 LT (Traditional Ligation) SV1 RED
W7 BL01 LT SV2 RED
W8 BL01 LT LV1
W9 BL01 LT LV2
W10 BL05 LT LV2
W11 Empty CMV/MV9
W12 BL01 LT SV3 NOT RED
W13 BL05 LT SV1
W14 BL05 LT SV2
Ladder

DNA[lanes 2-9*](lanes10-14) [8.0 μL] (4.0 μL)
10X buffer 1.5 μL
XbaI 1.0 μL
BsmbI 0.5 μL
dH2O [4μL] (8μL)
Total 15 μL --> 37°C/ 30 min.


  • DNA in lanes 2-9 have relatively low concentration (<90ng/μL).

Wells 6&7 (BL01 traditional ligation) look promising; will sequence tomorrow.
First four lanes are empty yet again yet have reasonable concentration and purity in the plate reader. Degradation?
Using DD123 & DD122 primers, a forward insertion will result in a band of length 3371 running the PCR reaction.


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