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Team:Evry/Notebook/Sensing/Phenol/08-18-2014
From 2014.igem.org
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<u> Dna extraction </u> <br> | <u> Dna extraction </u> <br> | ||
| - | Machery Nagel DNA purification Kit (<a href | + | Machery Nagel DNA purification Kit (<a href="http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NS.pdf">PROTOCOL</a>) |
<br> | <br> | ||
<u>PCR using VF2 and VR primer</u><br> | <u>PCR using VF2 and VR primer</u><br> | ||
Q5 polymerase <br> | Q5 polymerase <br> | ||
| - | <img src="https://static.igem.org/mediawiki/2014/3/ | + | Expected bands :<br> |
| + | DmpR: 2038 bp <br> | ||
| + | GFP B0031: 1331 bp <br> | ||
| + | GFP B0032: 1330 bp <br> | ||
| + | <u>Digestion</u> <br> | ||
| + | DmpR: SpeI&PstI<br> | ||
| + | GFP B0031/32: XbaI&PstI<br> | ||
| + | |||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2014/0/08/PCR_PRODUCT_VF2_VR_%2B_DIGESTION_PRODUCT_PNG.PNG"><img src="https://static.igem.org/mediawiki/2014/3/3a/1ST_CONSTRUCTION.PNG"><br><br> | ||
| + | <u>Analysis</u><br> | ||
| + | VF2/VR PCR products were in agreement with the expected size either for DmpR and GFP B0031/32.<br> | ||
| + | GFP 31/32 digestion products were in agreement with the expected size.<br> | ||
| + | However DmpR digestion revealed an unexpected profile.<br><br> | ||
| + | |||
| + | <u>Gel extraction</u><br> | ||
| + | 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.<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> | ||
| + | Machery Nagel kit (<a href="http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf">PROTOCOL 2</a>) | ||
| + | <br> | ||
| + | |||
</p> | </p> | ||
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)
