Team:BYU Provo/Team Thermosensor/The Beginning

From 2011.igem.org

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==Week Feb. 21 – 27==
 +
 +
* Feb. 22
 +
** Made PCR for thermosensor will check prouct tomorrow
 +
* Feb. 23
 +
** PCR products checked, thermosensor appears to have been amplified correctly
 +
** pPLAT extracted via qiaprep plasmid prep protocol
 +
 +
 +
==Week Feb. 28 – Mar. 6==
 +
 +
* Feb. 28
 +
** Phusion PCR for GFP in Thermosensor system
 +
*** ~35 uL ddH2O
 +
*** 10uL 5X phusion buffer
 +
*** 1.5uL 10mM dNTP’s
 +
*** 1uL each primer
 +
*** 1uL of diluted template DNA   
 +
*** 0.5 uL phusion polymerase
 +
** Purified PCR samples according to Qiagen Kit
 +
** Restriction Digest of insert and vector inserting thermosensor into pPLAT
 +
*** PCR product(insert) digestion (50uL rxn)
 +
**** ~14uL H2O
 +
**** 5uL 10X NEB buffer
 +
**** 0.5 uL 100X BSA
 +
**** 30uL DNA sample
 +
**** 1-2uL each restriction enzyme
 +
*** Vector Digestion (50uL rxn)
 +
**** ~14uL H2O
 +
**** 5uL 10X NEB buffer
 +
**** .5 uL 100X BSA
 +
**** 30uL DNA sample
 +
**** 1-2 uL each restriction enzyme
 +
***** Placed reactions in 37o bath for 1.5 hr+
 +
 +
* Mar. 2
 +
** Restriction Digests actually done today
 +
** Running a gel of purified thermoswitch and purified GFP
 +
*** (thermoswitch in 3rd well and GFP in 5th well)
 +
 +
* Mar. 4
 +
** Product from redo thermoswitch PCR
 +
 +
 +
==Week Mar. 21 – 27==
 +
 +
* Mar. 23
 +
** Low Melt gel ran on vector and insert, bands cut out and gel cut-outs melted at 65oC
 +
** Received pGLO from Dr. Grose on Monday to be transformed into competent cells to act as a positive control for GFP
 +
** pPLAT can be transformed into competent cells(DH5-a) as a neg. control
 +
*** (GFP was more widely used by the OxyR team so they did the transformation of pGLO into competent cells)
 +
** Began ligation for pPLAT and pBAD cut at BamHI and EcoRI
 +
*** 6.5 uL H2O
 +
*** 1.5uL 10X ligase buffer
 +
*** 1 uL T4 DNA ligase
 +
*** 3uL vector
 +
*** 3uL insert (didn’t put insert in control)
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Latest revision as of 02:20, 24 September 2011

Team BYU Provo

BYU Provo
 

Week Feb. 21 – 27

  • Feb. 22
    • Made PCR for thermosensor will check prouct tomorrow
  • Feb. 23
    • PCR products checked, thermosensor appears to have been amplified correctly
    • pPLAT extracted via qiaprep plasmid prep protocol


Week Feb. 28 – Mar. 6

  • Feb. 28
    • Phusion PCR for GFP in Thermosensor system
      • ~35 uL ddH2O
      • 10uL 5X phusion buffer
      • 1.5uL 10mM dNTP’s
      • 1uL each primer
      • 1uL of diluted template DNA
      • 0.5 uL phusion polymerase
    • Purified PCR samples according to Qiagen Kit
    • Restriction Digest of insert and vector inserting thermosensor into pPLAT
      • PCR product(insert) digestion (50uL rxn)
        • ~14uL H2O
        • 5uL 10X NEB buffer
        • 0.5 uL 100X BSA
        • 30uL DNA sample
        • 1-2uL each restriction enzyme
      • Vector Digestion (50uL rxn)
        • ~14uL H2O
        • 5uL 10X NEB buffer
        • .5 uL 100X BSA
        • 30uL DNA sample
        • 1-2 uL each restriction enzyme
          • Placed reactions in 37o bath for 1.5 hr+
  • Mar. 2
    • Restriction Digests actually done today
    • Running a gel of purified thermoswitch and purified GFP
      • (thermoswitch in 3rd well and GFP in 5th well)
  • Mar. 4
    • Product from redo thermoswitch PCR


Week Mar. 21 – 27

  • Mar. 23
    • Low Melt gel ran on vector and insert, bands cut out and gel cut-outs melted at 65oC
    • Received pGLO from Dr. Grose on Monday to be transformed into competent cells to act as a positive control for GFP
    • pPLAT can be transformed into competent cells(DH5-a) as a neg. control
      • (GFP was more widely used by the OxyR team so they did the transformation of pGLO into competent cells)
    • Began ligation for pPLAT and pBAD cut at BamHI and EcoRI
      • 6.5 uL H2O
      • 1.5uL 10X ligase buffer
      • 1 uL T4 DNA ligase
      • 3uL vector
      • 3uL insert (didn’t put insert in control)
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