Restriction Digests – Withering Syndrome Plasmds 20131211

Linearized three plasmids for use in WS qPCR standard curve NcoI. Each reaction contained the following:

Plasmid: 5uL

10x Buffer #3 (NEB): 5uL

H2O: 39uL

NcoI (NEB): 1uL

All reactions were incubated @ 37C for 1hr and then heat inactivated @ 65C for 20mins. 25uL of each reaction was run on a gel, along with 5uL of undigested plasmid to confirm digestion.

Results:

Gel Layout

  • Lane 1 – Hyperladder II (Bioline)
  • Lane 2 – pCR2.1/WS-1
  • Lane 3 – pCR2.1/WS-1 NcoI
  • Lane 4 – pCR2.1/WS-2
  • Lane 5 – pCR2.1/WS-2 NcoI
  • Lane 6 – pCR2.1/WS-3
  • Lane 7 – pCR2.1/WS-3 NcoI
  • Lane 8 – No template control

Ran the wrong ladder. However, we see complete digestion of pCR2.1/WS-1 and WS-2. pCR2.1/WS-3 may not be complete, as evidenced by the highest molecular weight band that is present in both the undigested and digested lanes. Although this has no effect on anything, pCR2.1/WS-3 has the insert in the opposite orientation as WS-1 and WS-2, which explains the different digestion pattern. Will create standard curves from all three digestions.

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