IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/22: Difference between revisions

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DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge
DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge


see QIAquick spin handbook:
see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]
http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2


===Innoculations===
===Innoculations===

Revision as of 11:42, 23 June 2010

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Images

Team Vector: HindIII + PstI digest of miniprepped V9-V12 (#5,6) to check whether insert is present.

Team Fence

GAL4DBD Miniprep

  • pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute
  • remaining overnight cell cultures were placed in fridge
  • decanted LB+amp, resuspended cells in 250 μL P1 buffer
  • contents transferred to eppendorfs
  • 250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
  • 350 μL N3 buffer added to each, tubes inverted 4-6 times
  • centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
  • centrifuged for 30-60 seconds, flow through discarded
  • 0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
  • QIAprep columns put into new eppendorfs
  • 50 μL buffer EB added to columns, let stand for 1 minute
  • centrifuged for 1 minute at 13,000 rpm
  • tubes labeled "GAL4" plus the specific colony number

PCR

PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)

1=95°C for 10:00 2=95°C for 00:15 3=50°C for 00:30 4=72°C for 01:30 5=steps 2-4 X 29 6=72°C for 10:00 7=4°C for ∞

PCR succesful

PCR for E10 LacI

  • above PCR was repeated without O2 so as to finalize our E10 biobrick for use
  • PCR was performed using the standard proportions of reagents
  • The above settings on the PCR machine were used

To Make Agarose Gel

  • 150 mL TAE buffer combined with 1.5 g agarose in beaker
  • solution microwaved until agarose disolved
  • 7.5 μL Ethidium Bromide added after beaker was no longer steaming
  • solution poured into clean gel mold and gel was left to solidify
  • E10 PCR product from the morning was consolidated into one tube,
  • final volume =90μL PCR product + 18μL 5X loading dye =110μL

DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge

see QIAquick spin handbook

Innoculations

Colonies from the following cultures were innoculated:

  • PMT413
  • PMT316
  • firefly luciferase
  • GFP (mut)
  • cre recombinase
  • lox66
  • lox71


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