Team:SCU China/Notebook

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-== iGEM&nbsp;SCU&nbsp;experiment&nbsp;protocol ==
-=== Transformation ===
-.&nbsp;Thaw&nbsp;the&nbsp;competent&nbsp;cells&nbsp;on&nbsp;ice&nbsp;for&nbsp;about&nbsp;10-15&nbsp;minutes.
-.&nbsp;Add&nbsp;50&nbsp;μL&nbsp;of&nbsp;thawed&nbsp;competent&nbsp;cells&nbsp;into&nbsp;each&nbsp;pre-chilled&nbsp;1.5mL&nbsp;eppendorf&nbsp;tube.
-.&nbsp;Add&nbsp;1.5&nbsp;μL&nbsp;of&nbsp;the&nbsp;resuspended&nbsp;DNA&nbsp;(or&nbsp;4&nbsp;μL&nbsp;of&nbsp;the&nbsp;ligation&nbsp;product)&nbsp;to&nbsp;the&nbsp;1.5&nbsp;mL&nbsp;tube,&nbsp;gently&nbsp;pipet&nbsp;up&nbsp;and&nbsp;down&nbsp;for&nbsp;a&nbsp;few&nbsp;times,&nbsp;make&nbsp;sure&nbsp;to&nbsp;keep&nbsp;the&nbsp;competent&nbsp;cells&nbsp;on&nbsp;ice.
-.&nbsp;Close&nbsp;the&nbsp;tubes&nbsp;and&nbsp;incubate&nbsp;the&nbsp;cells&nbsp;on&nbsp;ice&nbsp;for&nbsp;30&nbsp;minutes.
-.&nbsp;Heat&nbsp;shock&nbsp;the&nbsp;cells&nbsp;by&nbsp;immersion&nbsp;in&nbsp;a&nbsp;pre-heated&nbsp;water&nbsp;bath&nbsp;at&nbsp;42&nbsp;℃&nbsp;for&nbsp;60-90&nbsp;seconds.
-.&nbsp;Incubate&nbsp;the&nbsp;cells&nbsp;on&nbsp;ice&nbsp;for&nbsp;5&nbsp;minutes.
-.&nbsp;Add&nbsp;300&nbsp;μL&nbsp;of&nbsp;SOC&nbsp;(make&nbsp;sure&nbsp;that&nbsp;the&nbsp;broth&nbsp;does&nbsp;not&nbsp;contain&nbsp;antibiotics&nbsp;and&nbsp;is&nbsp;not&nbsp;contaminated)&nbsp;to&nbsp;each&nbsp;tube.
-.&nbsp;Incubate&nbsp;the&nbsp;cells&nbsp;at&nbsp;37&nbsp;℃&nbsp;for&nbsp;1-2&nbsp;hours&nbsp;while&nbsp;the&nbsp;tubes&nbsp;are&nbsp;rotating&nbsp;or&nbsp;shaking&nbsp;(The&nbsp;purpose&nbsp;of&nbsp;this&nbsp;step&nbsp;is&nbsp;to&nbsp;ensure&nbsp;the&nbsp;transformation&nbsp;efficiency).
-.&nbsp;Distribute&nbsp;200&nbsp;μL&nbsp;transformed&nbsp;cells&nbsp;uniformly&nbsp;over&nbsp;the&nbsp;appropriate&nbsp;LB&nbsp;plate&nbsp;(containing&nbsp;the&nbsp;proper&nbsp;antibiotics&nbsp;or&nbsp;not).
-.&nbsp;Incubate&nbsp;the&nbsp;plates&nbsp;at&nbsp;37&nbsp;℃&nbsp;for&nbsp;12-14&nbsp;hours,&nbsp;make&nbsp;sure&nbsp;the&nbsp;agar&nbsp;side&nbsp;of&nbsp;the&nbsp;plate&nbsp;is&nbsp;up.
-&nbsp;
-=== Liquid&nbsp;culture&nbsp;to&nbsp;grow&nbsp;bacteria&nbsp;from&nbsp;a&nbsp;single&nbsp;colony ===
-.&nbsp;Add&nbsp;5&nbsp;mL&nbsp;LB&nbsp;into&nbsp;each&nbsp;10&nbsp;mL&nbsp;or&nbsp;15&nbsp;mL&nbsp;tube.
-.&nbsp;Add&nbsp;proper&nbsp;antibiotics&nbsp;solution&nbsp;into&nbsp;each&nbsp;tube；
-.&nbsp;Pick&nbsp;a&nbsp;single&nbsp;colony&nbsp;on&nbsp;plates&nbsp;with&nbsp;tweezers&nbsp;clamping&nbsp;a&nbsp;pipette&nbsp;tip&nbsp;and&nbsp;then&nbsp;drop&nbsp;the&nbsp;tip&nbsp;containing&nbsp;transformed&nbsp;cells&nbsp;into&nbsp;appropriate&nbsp;LB&nbsp;containing&nbsp;proper&nbsp;antibiotics.&nbsp;
-.&nbsp;Seal&nbsp;tubes&nbsp;with&nbsp;sterile&nbsp;sealing&nbsp;film.
-.&nbsp;Incubate&nbsp;the&nbsp;cells&nbsp;at&nbsp;37&nbsp;℃&nbsp;for&nbsp;overnight&nbsp;while&nbsp;the&nbsp;tubes&nbsp;are&nbsp;rotating&nbsp;or&nbsp;shaking.
-&nbsp;
-=== 3A&nbsp;&nbsp;Assembly ===
-.&nbsp;Restriction&nbsp;Digests
-)&nbsp;The&nbsp;left&nbsp;part&nbsp;sample&nbsp;is&nbsp;cut&nbsp;out&nbsp;with&nbsp;EcoR&nbsp;I&nbsp;and&nbsp;Spe&nbsp;I.
-)&nbsp;The&nbsp;right&nbsp;part&nbsp;sample&nbsp;is&nbsp;cut&nbsp;out&nbsp;with&nbsp;Xba&nbsp;I&nbsp;and&nbsp;Pst&nbsp;I.
-)&nbsp;The&nbsp;linearized&nbsp;plasmid&nbsp;backbone,&nbsp;which&nbsp;is&nbsp;harvested&nbsp;by&nbsp;PCR，is&nbsp;a&nbsp;linear&nbsp;piece&nbsp;of&nbsp;DNA.&nbsp;It&nbsp;has&nbsp;a&nbsp;few&nbsp;bases&nbsp;beyond&nbsp;the&nbsp;EcoR&nbsp;I&nbsp;and&nbsp;Pst&nbsp;I&nbsp;restriction&nbsp;sites.&nbsp;It&nbsp;is&nbsp;cut&nbsp;with&nbsp;EcoR&nbsp;I&nbsp;and&nbsp;Pst&nbsp;I.
-.&nbsp;All&nbsp;3&nbsp;restriction&nbsp;digests&nbsp;are&nbsp;heated&nbsp;at&nbsp;80&nbsp;℃&nbsp;for&nbsp;20&nbsp;minutes&nbsp;in&nbsp;PCR&nbsp;meter&nbsp;to&nbsp;heat&nbsp;kill&nbsp;all&nbsp;of&nbsp;the&nbsp;restriction&nbsp;enzymes.
-.&nbsp;An&nbsp;equimolar&nbsp;quantity&nbsp;(relative&nbsp;quantity)&nbsp;of&nbsp;all&nbsp;3&nbsp;restriction&nbsp;digest&nbsp;products&nbsp;are&nbsp;combined&nbsp;in&nbsp;a&nbsp;ligation&nbsp;reaction.
-.&nbsp;The&nbsp;desired&nbsp;result&nbsp;is&nbsp;the&nbsp;left&nbsp;part&nbsp;sample's&nbsp;Spe&nbsp;I&nbsp;overhang&nbsp;ligated&nbsp;with&nbsp;the&nbsp;right&nbsp;part&nbsp;sample's&nbsp;Xba&nbsp;I&nbsp;overhang&nbsp;resulting&nbsp;in&nbsp;a&nbsp;scar&nbsp;that&nbsp;cannot&nbsp;be&nbsp;cut&nbsp;with&nbsp;any&nbsp;of&nbsp;our&nbsp;enzymes.
-.&nbsp;The&nbsp;new&nbsp;composite&nbsp;part&nbsp;sample&nbsp;is&nbsp;ligated&nbsp;into&nbsp;the&nbsp;construction&nbsp;plasmid&nbsp;backbone&nbsp;at&nbsp;the&nbsp;EcoR&nbsp;I&nbsp;and&nbsp;Pst&nbsp;I&nbsp;sites.
-.&nbsp;When&nbsp;the&nbsp;ligation&nbsp;is&nbsp;transformed&nbsp;into&nbsp;cells&nbsp;and&nbsp;grown&nbsp;on&nbsp;plates&nbsp;with&nbsp;antibiotic&nbsp;C,&nbsp;only&nbsp;colonies&nbsp;with&nbsp;the&nbsp;correct&nbsp;construction&nbsp;are&nbsp;survived.
-&nbsp;
-'''Traditional&nbsp;assembly&nbsp;(standard&nbsp;assembly)&nbsp;methods'''
-For&nbsp;Standard&nbsp;Assembly,&nbsp;a&nbsp;part&nbsp;sample&nbsp;is&nbsp;cut&nbsp;out&nbsp;from&nbsp;its&nbsp;plasmid&nbsp;backbone&nbsp;and&nbsp;inserted&nbsp;into&nbsp;the&nbsp;prefix&nbsp;of&nbsp;a&nbsp;plasmid&nbsp;backbone&nbsp;of&nbsp;another&nbsp;part&nbsp;(we&nbsp;use&nbsp;EcoRⅠand&nbsp;Xba&nbsp;Ӏ&nbsp;to&nbsp;digest&nbsp;plasmid&nbsp;backbone&nbsp;and&nbsp;use&nbsp;EcoRⅠand&nbsp;SpeⅠto&nbsp;digest&nbsp;the&nbsp;target&nbsp;part).&nbsp;Two&nbsp;restriction&nbsp;digests&nbsp;are&nbsp;done,&nbsp;one&nbsp;for&nbsp;the&nbsp;part&nbsp;sample&nbsp;that&nbsp;will&nbsp;be&nbsp;moved&nbsp;and&nbsp;one&nbsp;for&nbsp;the&nbsp;plasmid&nbsp;backbone&nbsp;that&nbsp;will&nbsp;receive&nbsp;it.&nbsp;The&nbsp;digests&nbsp;are&nbsp;then&nbsp;run&nbsp;on&nbsp;a&nbsp;gel&nbsp;and&nbsp;using&nbsp;gel&nbsp;purification&nbsp;the&nbsp;required&nbsp;fragments&nbsp;are&nbsp;isolated&nbsp;(the&nbsp;part&nbsp;sample&nbsp;and&nbsp;the&nbsp;cut&nbsp;plasmid&nbsp;backbone).&nbsp;The&nbsp;purified&nbsp;insert&nbsp;and&nbsp;cut&nbsp;plasmid&nbsp;backbone&nbsp;are&nbsp;ligated&nbsp;and&nbsp;the&nbsp;resulting&nbsp;composite&nbsp;part&nbsp;can&nbsp;be&nbsp;transformed&nbsp;into&nbsp;E.&nbsp;coli&nbsp;cells.
-&nbsp;
-'''Miniprep&nbsp;protocol'''
-&nbsp;&nbsp;&nbsp;We&nbsp;use&nbsp;MINIPREP&nbsp;PLASMID&nbsp;KIT&nbsp;(TIANGEN&nbsp;BIOTECH,&nbsp;CHINA)&nbsp;for&nbsp;miniprep.
-&nbsp;
-'''Restriction&nbsp;digestion'''
-&nbsp;&nbsp;&nbsp;We&nbsp;conduct&nbsp;our&nbsp;experiments&nbsp;following&nbsp;the&nbsp;standard&nbsp;protocol&nbsp;from&nbsp;the&nbsp;HD,&nbsp;except&nbsp;for&nbsp;no&nbsp;adding&nbsp;BSA&nbsp;into&nbsp;our&nbsp;mixture.
-&nbsp;
-'''Ligation'''
-&nbsp;&nbsp;&nbsp;We&nbsp;follow&nbsp;the&nbsp;standard&nbsp;protocol&nbsp;from&nbsp;HD&nbsp;web&nbsp;to&nbsp;conduct&nbsp;our&nbsp;ligation&nbsp;experiments.
-&nbsp;
-'''Making&nbsp;linearized&nbsp;plasmid&nbsp;backbones'''
-&nbsp;&nbsp;&nbsp;We&nbsp;do&nbsp;this&nbsp;by&nbsp;the&nbsp;bulk&nbsp;production&nbsp;protocol&nbsp;from&nbsp;HD.
-&nbsp;
-'''Competent&nbsp;cells'''
-&nbsp;&nbsp;&nbsp;We&nbsp;choose&nbsp;DH5α&nbsp;from&nbsp;TIANGEN&nbsp;BIOTECH&nbsp;as&nbsp;our&nbsp;competent&nbsp;cells&nbsp;meterials.
-&nbsp;
-'''Quantifying&nbsp;fluorescence&nbsp;of&nbsp;GFP&nbsp;expressed&nbsp;in&nbsp;E.&nbsp;coli'''
-We&nbsp;choose&nbsp;plate&nbsp;reader&nbsp;to&nbsp;detect&nbsp;fluorescence&nbsp;of&nbsp;GFP&nbsp;expressed&nbsp;in&nbsp;E.&nbsp;coli.
-Before&nbsp;detecting&nbsp;the&nbsp;fluorescence&nbsp;on&nbsp;plate&nbsp;reader,&nbsp;we&nbsp;view&nbsp;the&nbsp;fluorescence&nbsp;of&nbsp;E.&nbsp;coli&nbsp;with&nbsp;a&nbsp;fluorescence&nbsp;microscope&nbsp;to&nbsp;make&nbsp;sure&nbsp;that&nbsp;GPF&nbsp;expresses&nbsp;normally&nbsp;in&nbsp;vivo.&nbsp;
-Incubate&nbsp;bacteria&nbsp;for&nbsp;certain&nbsp;periods&nbsp;of&nbsp;time,&nbsp;then&nbsp;take&nbsp;some&nbsp;sample&nbsp;out&nbsp;and&nbsp;add&nbsp;it&nbsp;into&nbsp;each&nbsp;well&nbsp;on&nbsp;96&nbsp;well&nbsp;plate&nbsp;(for&nbsp;our&nbsp;experiment,&nbsp;we&nbsp;add&nbsp;150&nbsp;μL&nbsp;per&nbsp;well).
-</div>|}
 |}
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