Chloroform Cleanup – EcoRI-digested Withering Syndrome Phage DNA from earlier today

To concentrate the sample, a chloroform cleanup and EtOH precipitation was performed. An equal volume of chloroform was added to the sample (220uL), vortexed for 30s and spun @ 16,000g at 4C for 15mins. The aqueous phase was transferred to a clean tube for EtOH precipitation.

0.1 volumes (16.8uL) of 3M NaOAC (sodium acetate; pH = 5.2) and 2.5 volumes (462uL) of ice cold 100% EtOH were added to the sample and mixed thoroughly. The sample was incubated O/N at -20C. The following day the sample was pelleted by spinning at 16,000g at 4C for 30mins. As expected, there was no visible pellet due to the extremely low quantity of DNA in the sample. The supernatant was discarded, the pellet was gently washed (no mixing/vortexing) with 70% EtOH, and spun @ 16,000g at 4C for 15mins. The supernatant was discarded the pellet was air dried for 15mins. The sample was reconstituted in 10uL of Buffer EB (Qiagen), which is simply 10mM Tris-HCl, and stored at 4C.

Gel Purification – EcoRI-digested pCR2.1 Vector from earlier today

Gel-excised band from earlier today was purified using the Ultra DA-free (Millipore) spin column according to the manufacturer’s protocol. Purified DNA was stored at 4C.

Restriction Digestions – Withering Syndrome Phage DNA and p16RK3

Performed restriction digestions on Phage RLO DNA (isolated on 20121130) and p16RK3 using EcoRI from NEB.

Phage RLO Digest

  • DNA – 192uL
  • 10x EcoRI Buffer – 22uL
  • EcoRI – 4uL
  • H2O – 2uL
  • TOTAL = 220uL

p16RK3 Digest

  • DNA (1ug) – 5.39uL
  • 10x EcoRI Buffer – 5uL
  • EcoRI – 1uL
  • H2O – 38.61uL
  • TOTAL = 50uL

Samples were incubated at 37C for 1hr. The p16RK3 sample was run on a 0.8% TBE gel to confirm digestion and isolate the vector-only band from the sample. The Phage RLO sample was subject to a chloroform cleanup and EtOH precipitation.

Results:

Digestion of Phage RLO DNA was not verified due to the extremely low quantities of DNA. In order to minimize loss of material, we will trust that the digestion was successful if the digestion of p6RK3 was successful.

Gel Loading

Lane 1 – Hyperladder I (Bioline)

Lane 2 – p16RK3 EcoRI

Neglected to run undigested p16RK3. However, the digestion pattern looks correct. The empty vector (pCR2.1; Invitrogen) should be 3931bp and we see the largest molecular weight band of the digest is running at ~4000bp. That band was excised and will be purified for subsequent ligation.