User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/16

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(Bench work)
Current revision (14:26, 17 August 2011) (view source)
(Objective)
 
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==Objective==
==Objective==
-
* Run QuikChange on pMXB10 from [[yesterday]](by Dr. Muratore) on gel
+
* Run QuikChange on pMXB10 from [[../15#Bench work|yesterday]](by Dr. Muratore) on gel
* Check transformations on plates
* Check transformations on plates
* Prepare for expression of pMAL-pIII and pMXB10/start cultures from colonies on plates
* Prepare for expression of pMAL-pIII and pMXB10/start cultures from colonies on plates

Current revision

AU CHEM-570 Lab Prep Main project page
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Contents

Objective

  • Run QuikChange on pMXB10 from yesterday(by Dr. Muratore) on gel
  • Check transformations on plates
  • Prepare for expression of pMAL-pIII and pMXB10/start cultures from colonies on plates

Bench work

  1. Ran 1.0% agarose DNA gel with 2-stage QuikChange of pMXB10 for +His tag from yesterday(by Dr. Muratore)
    • Lane 1: Ladder
    • Lane 2: ----
    • Lane 3: ----
    • Lane 4: pMXB10 QC +His 8/15
    • Lane 5: ----
    • Lane 6: ----
    • Lane 7: pMXB10 QC Ctrl 8/15
  2. Due to perceived failure of QuikChange from yesterday, performed a test PCR with pCMV-SPORT6+BSA to amplify BSA gene
    • Vary Pfu Turbo buffer and Pfu Turbo polymerase in PCR reaction:
    • Buffer A + Polymerase 1 == 'A1'
    • Buffer A + Polymerase 2 == 'A2'
    • Buffer B + Polymerase 1 == 'B1'
    • Buffer B + Polymerase 2 == 'B2'
    • Follow same protocol as 5/26
  3. Took 1 colony from each of pMAL-pIII and pMXB10 in DH10B and put into 10mL of LB medium with 10μL of 100mg/mL ampicillin
    • Grew O/N at 37°C
  4. Took 1 colony from each of pMAL-PIII and pMXB10 in ER2566 and put into 50mL of LB medium with 50μL of 100mg/mL ampicillin
    • Grew O/N at 37°C
  5. Replated the four lowest-yielding transformations:
    • pMAL-pIII and pMXB10 in ER2566
    • BamHI-digested pKK223-3 ligations of 10μL and 15μL in DH10B

Results

Gel

Image: DNA_gel_pMXB10_8.16.2011_labeled.jpg

    • Did not see what we wanted on gel -- suspect issue with reagents
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