Haynes Lab:Notebook/Engineering PC-TFs/2015/05/18

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  • Restriction Digest/Gel Verification, Concentration Reading

Digest and Gel Verification

Reagent Volume

V0120 [no cut sites]
3200 bp

pUC57 [KpnI]
Linearize: 2900 bp


20 μL/lane; 1% agarose;
Observed lengths:
W1: Ladder
W2: KAH 021 w/ enzyme
W3: KAH 021 w/o enzyme
W4: KAH 022 w/ enzyme
W5: KAH 022 w/o enzyme
W6: KAH 023 w/ enzyme
W7: KAH 023 w/o enzyme
W8: KAH 024 w/ enzyme
W9: KAH 024 w/o enzyme


DNA 5.0 μL 5.0 μL 8.0 μL 8.0 μL 5.0 μL 5.0 μL 5.0 μL 5.0 μL (From ladder 2 to ladder 9)
10X buffer 2.0 μL (for all)
KpnI 1.0 μL (for even numbered wells only)
dH2O 8.5 μL 8.5 μL 9.5 μL 9.5 μL 10.5 μL 10.5 μL
Total 20 μL --> 37°C/ 15 min.

Note: This experiment is to verify what backbone the linker is in. There are two wells for each linker, one contains the restriction enzyme KpnI while the other does not. V0120 does not have a cut site for KpnI. If the two wells look the same, the linker is in V0120. If the two wells look different, the linker is in pUC57. As seen in the image, all the wells without the enzyme ran through the gel faster than all the wells with this enzyme. This indicates that it is a supercoiled plasmid as plasmids run faster than linear DNA.