Team:BYU Provo/Team Thermosensor/Week6
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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)
- LacZ PCR cleanup
- 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.
- Digested GFP at the HindIII and XbaI sites
- 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
- Restriction Digest of LacZ and GFP
- 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.