Restriction Digests – Withering Syndrome Clone Plasmids from 20120718

Performed restriction digest on all four clones using NcoI. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.

Each reaction contained:

Plasmid – 5uL

10x Buffer – 5uL

NcoI – 1uL

H2O – 39uL

After inactivation, 5uL from each reaction were run on a 1% TBE gel to confirm digestion. 1uL of undigested plasmid was run along side the corresponding digest.

The four clones are referred to as:

  • pWC8
  • p16RK3
  • p16RK7
  • p18RK7

Results:

Gel Loading (left to right):

Lane 1 – Hyperladder I (Bioline)

Lane 2 – pWC8 (Und.)

Lane 3 – pWC8 (NcoI)

Lane 4 – p16RK3 (Und.)

Lane 5 – p16RK3 (NcoI)

Lane 6 – p16RK7 (Und.)

Lane 7 – p16RK7 (NcoI

Lane 8 – p18RK7 (Und.)

Lane 9 – p18RK7 (NcoI)

Lane 10 – Hyperladder I (Bioline)

All digests are complete. All clones reveal the same restriction digestion pattern, producing two bands: ~2500bp and ~3000bp. The band sizes total ~5500bp, which is in line (5432bp) with the full withering syndrome 16s clone in the pCR2.1 TOPO vector (Invitrogen). Will quant and prepare a dilution series for qPCR.

One thought on “Restriction Digests – Withering Syndrome Clone Plasmids from 20120718

  1. Pingback: Sample Prep – Withering Syndrome p18RK7 Plasmid Standard Curve Dilution | Sam's Notebook

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