User:Eric M. Walters/Notebook/Spring 2012/2012/05/02

From OpenWetWare

Jump to: navigation, search
Cyanobacterial acrylate production Main project page
Previous entry      Next entry

Screening putative acsA::accBCDA clones

  • Many small colonies arose from the streakouts done Monday of the two transformant colonies
  • Will be screening both of them with the original (now outside of the insertion) primers acsA-upF and -dnR
  • Controls are gDNA (should yield just the acsA + 2x500 bp flanking regions - 3.2 kb total), and a ΔacsA 7002 gDNA tube I found in Matt's freezer. He's not in right now, but this should let me make sure that the process of deleting acsA hasn't disrupted the near-outside primers I'm using. However, I don't yet know what size I'm expecting to see.

Colony PCR reactions and conditions

  • 20 μL reactions:
    • 10 μL GoTaq
    • 1 μL acsA-upF
    • 1 μL acsA-dnR
    • Either 1 μL gDNA, or pick of colony
    • 7 μL H2O
  • PCR cycle:
    • 95°C - 2:00
    • 95°C - 0:30
    • 61°C - 0:30
    • 72°C - 5:40 (5.6 kb at 1 kb/min)
    • Repeat 30x
    • 72°C - 5:00


Gel after 45 minutes at 100V:

Image:Pflegerlab 2012-05-02 17hr 44min acsA;;accBCDA colony PCR (45 minute).jpg


Personal tools