Team:EPF-Lausanne/Notebook/3 August 2012

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| Control of NotI w/ PMP2||Control of SpeI w/ PMP2||||eGFP pcr product||TNFR pcr product||SEAP pcr product||||
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Revision as of 14:17, 6 August 2012



Digestion of PMP and PGL4.30 for ligation

prepared PMP2 , PGL2 and PGL3 for ligation

restriction enzyme digest mixtures were as follows:

PMP2 control for plasmid

  • DNA 2µL
  • HindIII 1µL
  • XbaI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 40.5 µL

PMP2 control for NotI

  • DNA 2µL
  • NotI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 41.5 µL

PMP2 control for SpeI

  • DNA 2µL
  • SpeI 1µL
  • N4 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 41.5 µL


PMP2 for ligation

  • DNA 19µL
  • SpeI 1µL
  • NotI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 23.5 µL

PGL2 for ligation

  • DNA 12µL
  • HindIII 1µL
  • MfeI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 31 µL

PGL3 for ligation

  • DNA 16µL
  • HindIII 1µL
  • FseI 1µL
  • N2 buffer 10x 5µL
  • BSA 100x 0.5µL
  • Water 26.5 µL

All let 1:30 hours at 37ºC, shaking (600 rpm).


Bands got in the control of DNA and enzymes. Everything seems to be correct now!

After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).


Gel with digested backbones and pcr products for readouts


PMP2 cut w/ NotI SpeI ' PGL2 cut w/ HindII MfeI ' PGL3 cut w/ HindII FseI ' control of HindII w/ PGL3 control of FseI w/ PGL3
Control of NotI w/ PMP2Control of SpeI w/ PMP2eGFP pcr productTNFR pcr productSEAP pcr product



Protocol: None

Forgot to insert protocol.


Comments

Insert comments about what happened.


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