8th June 2015
Yi Han
• Miniprep of gfp plasmids, preparation of samples for sequencin
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9th June 2015
Yi Han
• All gfp plasmids had a correct sequence
• Yun Ting kept glycerol stocok for all, and inoculation of 3mL of gfp3 into 100mL LB+amp for midiprep
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10th June 2015
Yi Han & Yun Ting
• Midiprep of gfp plasmid
• PCR of gfp with KpnI-gfp and XhoI-gfp primers
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11th June 2015
Yun Ting & Duy
• Nanodrop of gfp product → 595.5ng/ul
• RE digest of EsaR vector with KpnI/XHoI
• Gel electrophoreseis at 1000V for 30min
• Gel extraction: 3.9ng/ul and 6.9ng/ul for Esa fragment and GFP --> low yield
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12th June 2015
Yun Ting
• Gel extraction using Promega binding solution to melt gel, followed by thermo scientific kit
Adrian
• Gel extraction optimisation
• Hypothesised that the Binding buffer has a problem/DNA does not bind to column
1: 2X promega binding buffer volume
2: Increase incubation time for binding to 5min
• Switch binding buffer to that of thermo scientific PCR purification kit
• Use sodium acetate if available? To facilitate stronger binding to column
Results:
1: ~10ng/ul
2: ~9ng/ul
•Further optimisation-> warm buffer, incubate for 5min
•Elute in 30/20ul smaller volumes
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13th June 2015
Yi Han
1: Thermoscientific miniprep columns with 2XThermoscientific binding buffer
2: Thermoscientific PCR purification kit 2X buffer
3: Promega kit 2X buffer
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