IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/07/01: Difference between revisions

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  10X NEB buffer.. 2μL
  10X NEB buffer.. 2μL
  dNTPs..........0.5μL
  dNTPs..........0.5μL
DNA............0.5μL
  Primer 1(5μM)... 2μL
  Primer 1(5μM)... 2μL
  Primer 2(5μM)... 2μL
  Primer 2(5μM)... 2μL
ddH2O........... 2μL
  Polymerase.......1μL
  Polymerase.......1μL
   
   

Revision as of 09:39, 10 September 2009

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July 1, 2009

PCR

  • ran a gel of yesterday's PCR (62°C, magnesium gradient)
  • Gel is mostly blank except for 035
  • Looks like we didn't add polymerase and primers, but we certainly did
  • Possibly the change in temperature messed things up?
  • Will use 035C(2) in restriction digest to try to determine nature of the band

Restriction Digest

  • BstBI/BglII double digest
  • Should have 1 fragment at 333bp if we have GFP - AHI construct OR kan-ADH construct
  • Ran a gel of product. Weird: No apparent digest.

Gel Purification

  • PCR. Used purified product as template.
  • 2 MgCl gradients (0 and 2) at 58°C annealing
  • for 49, will try all 4 gradients (0, 1, 2, 4) at 55°C annealing
  • After running PCR, realized we mixed up primers/templates for 139 and 186; they were discarded.
  • Ran a gel; only got funky bands at 850kb; no actual product
Top: Ladder, -DNA, -Pol, 035, -, 49, 213, -, 236, 346. At 55C 41(0), 49(1), 49(2), 49(4)
Bottom: Ladder, -, -, 035, 49, 186, 213, 236, 346
  • must be a mispriming error, which is weird because they are supposedly no redundancies according to the sequence

PCR

  • Realized (after running out of polymerase) that we've been doing our reactions all wrong. Correct recipe is below. Will have to order more polymerase and/or use another kind.
  • Correct PCR recipe using Accusure polymerase (20μL RXN)
10μL of reaction mix
10X NEB buffer.. 2μL
dNTPs..........0.5μL
DNA............0.5μL
Primer 1(5μM)... 2μL
Primer 2(5μM)... 2μL
ddH2O........... 2μL
Polymerase.......1μL

10μL of H2O/MgCl mix (concentrations of MgCl at 0, 1, 2 4). Use 20mM MgCl 

 MgCl......ddH2O
0: 0.......10μL
1: 1........9μL
2: 2........8μL
4: 4........6μL

*Add reaction mix to H2O/MgCl mix at whatever MgCl concentration you desire. Total will be 20μL




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