Team:Macquarie Australia/Notebook3

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PROJECT LAB BOOK


Welcome to the Macquarie University project lab book page!

DAY-BY-DAY PROGRESS FOR THE TRANSFORMATION AND CLONING

22nd September 2010

Plasmid Prep using Qiagen Plasmid Midi Prep Kit

  • The pET-3A vector plasmid that is to be used for the transformation needs to be purified
  • The pET-3A plasmid was purified using the Qiagen Plasmid Midi Prep Kit as per the manufacturer’s protocols
  • The only difference made to the protocol is that only 5ml of Buffer QC was used on the 2nd wash step
  • The plasmid (two samples A & B) as well as some column flow-through collected at different stages of the purification were run on a GelRed post-stained 1.5% agarose gel and photo taken for visualization (see below).
  • A NanoDrop spectrophotometer reading was also recorded to check the quality of the extracted genomic DNA.
  • There was no DNA pellet formed at the end of this protocol so we may have lost some DNA
  • There was also some plasmid observed in some of the column flow through so some plasmid has been lost
  • Figure 10. Qiagen Plasmid Midi Prep results:

    Figure 10. GelRed post-stained 1.5% agarose gel of pET-3A Plasmid purification. In lanes 1, 4, 5 and 15 there is a 1kb Molecular ladder. In lane 2 there is the Plasmid purification sample A and in lane 3 is the Plasmid purification sample B. In lanes 6 to 14 there are flow-throughs that were collected at different stages throughout the protocol. There is some DNA visible in these lanes so some of the DNA has been lost. Possibly genomic DNA they are quite high (didn’t move through the wells)