Team:BYU Provo/Team Thermosensor/Week6

Week 6 (May 22 - 28)

• May 23 LacZ ran on gel-product YES!
  • LacZ PCR cleanup
    • Cleaned up LacZ according to Qiaquick protocol and labeled as T 20-1(needs to be checked on gel)
• May 25
  • Restriction Digest of LacZ and GFP
    • Digested GFP at the HindIII and XbaI sites
      • oops...we realized that our reverse LacZ primer had a BglII site in it when we needed an XbaI site. Dr. Grose ordered a new primer with an XbaI restriction site in it.
  • Confirming pBAD clones by restriction digest
    • 5uL buffer 4
    • 0.5 uL BSA
    • 30 uL plasmid(pIG 17-4 and 17-5 each in individual tubes)
    • 12uL H2O
    • THEN MIX WELL AND ADD:
    • 1.5 uL BamHI-HF
    • 1.5 uL EcoRI-HF
      • If the gel shows two bands then pBAD was inserted successfully into pIG17-4 and/or pIG17-5. The Digest of pIG174 will be labeled as RD 20-4 and the digest of pIG17-5 will be labeled RD 20-5.
      • placed RDs in 37oC bath overnight and placed in the freezer
• May 27
  • Running RD 20-4 and RD 20-5 on an agarose gel. We will be using a digested pBAD gene and pPLAT digested at BamHI and PstI as our controls on the gel.