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Team:Evry/Notebook/Sensing/Phenol/08-18-2014
From 2014.igem.org
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<u>Gel extraction</u><br> | <u>Gel extraction</u><br> | ||
| - | Extraction of GFP B0031/32 digestion product [XbaI-PstI] | + | Extraction of GFP B0031/32 digestion product [XbaI-PstI]<br> |
| - | After confirmation of relevant electrophoresis bands, cut the gel all around the band and as close as possible to it. | + | After confirmation of relevant electrophoresis bands, cut the gel all around the band and as close as possible to it.<br> |
Place the resulting piece into a 2ml microcentrifuge tube. Add 1µl of Binding buffer to 1 µg of gel. Place the tube at 55°C to 60°C during 10min or until gel turn completely liquid.<br> | Place the resulting piece into a 2ml microcentrifuge tube. Add 1µl of Binding buffer to 1 µg of gel. Place the tube at 55°C to 60°C during 10min or until gel turn completely liquid.<br> | ||
Follow classical DNA purification protocol.<br> | Follow classical DNA purification protocol.<br> | ||
Latest revision as of 22:18, 15 September 2014
Dna extraction
Machery Nagel DNA purification Kit (PROTOCOL)
PCR using VF2 and VR primer
Q5 polymerase
Expected bands :
DmpR: 2038 bp
GFP B0031: 1331 bp
GFP B0032: 1330 bp
Digestion
DmpR: SpeI&PstI
GFP B0031/32: XbaI&PstI
Analysis
VF2/VR PCR products were in agreement with the expected size either for DmpR and GFP B0031/32.
GFP 31/32 digestion products were in agreement with the expected size.
However DmpR digestion revealed an unexpected profile.
Gel extraction
Extraction of GFP B0031/32 digestion product [XbaI-PstI]
After confirmation of relevant electrophoresis bands, cut the gel all around the band and as close as possible to it.
Place the resulting piece into a 2ml microcentrifuge tube. Add 1µl of Binding buffer to 1 µg of gel. Place the tube at 55°C to 60°C during 10min or until gel turn completely liquid.
Follow classical DNA purification protocol.
Machery Nagel kit (PROTOCOL 2)
