====Digestion of GFP 1.9 PCR product from the 9th September====
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''By Maxence & Victor''
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As we had several issues to clones GFP 1.9 in pSB1C3 by Gibson and digestion-ligation, GFP 1.9 PCR product from the 9th September was digested by restriction NdeI enzymes in order to verify if the template we used was the good one.
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For that purpose, GFP 1.9 PCR product from the 9th September was cut by restriction enzymes NdeI as following:
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* 2 µL of GFP 1.9 PCR product from the 9th September
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* 1 µL of buffer orange
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* 1 µL of restriction enzyme NdeI
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* 6 µL of water
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The mix was incubated for 30 minutes at 37°C. Then, 20 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
====Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation====
====Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation====
''By Maxence & Victor''
''By Maxence & Victor''
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PRECIPITATION ETOH
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Once template and vector were cut, they were mix together with water and were precipitated by ethanol as they were not concentrated enough. The ethanol precipitation was run as following:
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* 28 µL of template (pPS16_019 treated by XbaI & PstI)
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* 13 µL of vector (pPS16_018 treated by SpeI & PstI)
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* 9 µL of water
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* 50 µL of isopropanol
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* 5 µL of CH<inf>3</inf>COONa
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Once template and vector were cut, DNA ligase was used to join the sticky ends of the template and vector together:
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DNA ligase was used to join the sticky ends of the template and vector together:
