Tag Archives: QIAprep

Plasmid Isolation & Sequencing – C.gigas COX2/PGS2 Clones (from yesterday)

Isolated plasmid DNA from 3mL of liquid cultures that were inoculated yesterday using Qiagen’s miniprep kit. DNA was eluted with 50uL of EB. DNA was prepped and sent for sequencing to ASU sequencing facility. Each clone was sequenced two times in each direction. Samples are as follows:

Name – Clone # Primer

  • SJW01 – 1 M13F
  • SJW02 – 1 M13F
  • SJW03 – 1 M13R
  • SJW04 – 1 M13R
  • SJW05 – 2 M13F
  • SJW06 – 2 M13F
  • SJW07 – 2 M13R
  • SJW08 – 2 M13R
  • SJW09 – 3 M13F
  • SJW10 – 3 M13F
  • SJW11 – 3 M13R
  • SJW12 – 3 M13R
  • SJW13 – 4 M13F
  • SJW14 – 4 M13F
  • SJW15 – 4 M13R
  • SJW16 – 4 M13R

Clone #s are as follows:

1 – 5′ Library Top band

2 – 5′ Library Mid band

3 – 5′ Library Bottom band

4 – 3′ Library band

Results:

Sequencing results received 20110801. SJW15 and 16 apparently stop abruptly. The sequencing facility believes this to be caused by secondary structure of the template. Depending on how things align, I may consider using 7-daeza-GTP in a PCR reaction and re-sequencing this clone, as the 7-daeza-GTP helps relax secondary structure.

Spoke with Steven and he suggested just designing new primers closer to each other and resubmit.

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