Haynes Lab:Notebook/Engineering PC-TFs/2015/04/06

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Summary

Summary

  • Restriction Digest/Gel Verification, Concentration Reading

Digest and Gel Verification

Reagent Volume

Expected:
BL01 Ligation Colonies [XbaI/ApaLI]
Forward insertion: 255, 288, 883, 1243, 2037, 3017
Reverse insertion: 288, 255, 1122, 1243, 2037, 2783
No insertion: 268, 883, 1243, 2783

BL09 Ligation Colonies [XbaI/ApaLI]
Forward insertion: 255, 288, 883, 1243, 3977
Reverse insertion: 255, 288, 1243, 2082, 2783
No insertion: 268, 883, 1243, 2783


20 μL/lane; 1% agarose;
Observed lengths:
W1: Ladder
W2: Bl09 TL V1
W3: Bl09 TL V2
W4: BL01 LSR V1
W5: BL01 LSR V2
W6: BL01 LSR V3
W7: BL01 LSR V4

Ladder

DNA 4.0 μL 4.0 μL 4.0 μL 3.0 μL 2.0 μL 2.0 μL (From ladder 2 to ladder 6)
10X buffer 2.0 μL (for all)
XbaI 1.0 μL (for all)
ApaLI 1.0 μL
dH2O 12 μL 12 μL 12 μL 13 μL 14 μL 14 μL
Total 20 μL --> 37°C/ 45 min.


Note: Wells 2 and 3 show the same bands (Bl09). Bands are around 4000, 1300, 1000, and 300. These lengths are for forward insertion so the transformation was successful.Well 3 shows more higher lengths than the other 3 wells (all are Bl01). Well 5 does not have the unknown band around 4000-5000. Well 4 contains the bands around 2000, 3000, 1200, 1000, and 200. It has an unknown band around 5000. Well 5 contains the bands around 2000, 3000, 1500, 1000, and 200 so this transformation was successful. Wells 6 and 7 show a hide band around 5000. They show a band around 1200, 1000, and 200. No bands around 2000 or 3000 can be seen clearly which doesn't support any of the scenarios. Reactions were incubated for 45 minutes because the gel had to be remade. Electrophoresis flashed E1. There was less volume of Bl01 LSR V1



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