Team:LCG-UNAM-Mexico:Journals:Uriel
From 2009.igem.org
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DH5alpha competent cells were transformed and platted over LB Amp100 and incubate at 37ºC | DH5alpha competent cells were transformed and platted over LB Amp100 and incubate at 37ºC | ||
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+ | == September 28, 2009 == | ||
+ | |||
+ | Controls were inconsistent in particular the re-ligation of dephosphorylated plasmid which gave | ||
+ | the same amount of colonies compared with ligation of cox+ogr+ter and 12 so we are going to | ||
+ | re-dephosphorylate the vector 12 and perform a new ligation reaction. | ||
+ | |||
+ | Dephosphorylation Reaction: | ||
+ | Plasmid 10µL | ||
+ | Buffer 3µL | ||
+ | Enzyme 1µL | ||
+ | Water 16µL | ||
+ | Total 30µL | ||
+ | |||
+ | Incubation | ||
+ | 1)10 min --> 25ºC | ||
+ | 2)20 min --> 65ºC | ||
+ | |||
+ | A 1% agarose gel was run to see the concetration of the plasmid and the insert, this to put | ||
+ | the right amounts of insert and plasmid. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | The samples that we are looking on the gel correspon to cox+ogr+ter+18 digested with XbaI/SpeI and 12 | ||
+ | Once we checked concetrations we can do the ligation reaction. | ||
+ | Ligation Reaction: | ||
+ | |||
+ | T4 ligase 1µL | ||
+ | Buffer 2µL | ||
+ | cox+ofr+ter 12µL | ||
+ | 12 4µL | ||
+ | Water 1µL | ||
+ | Total 30µL | ||
Revision as of 21:23, 20 October 2009