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From 2013.igem.org

{ "date" : "2013-08-10", "author" : "viola-michele-emil", "title" : "Bacillus will not win!!!", "content" : "Today Michele joined the Bacillus' team. We redid 20 ml of fresh transformation media following the Groningen's protocol. Then we did 4 inocula and we waited until they reached the OD 1.00. During this time we performed a digestion with ScaI HF enzyme to tranform pXyl+EFE+B0015 in Bacillus. After an hour of digestion of three different quantities of DNA

we deactivate the digestion enzyme 20 minutes at 80°, the we added SAP for an hour at 37° and also this was deactivated for 20 minutes at 80°.It was also done an electrophoretic run to verify the result of this digestion: As you can see from the picture there is a unique band at about 6000 bp (Kapa Universal Ladder was used). We think that the band in the ladder at 5000 bp is not present because it remains under the bigger lane at 4000. When one of the 2 ml inocula reached the right OD we splitted it into 5 aliquotes of 400µl tubes and we transformed the three pXyl+EFE+B0015 digestions and two quantities of pSpac+EFE+B0015... tomorrow we will see. Unfortunately The first column(the Emil's one) shows two bands, it means that there are two restriction site for Sca1.This fact is possible only if the GFP isn't the E0840 but the E0240,probably they were mistaken the last year by the previus Igem team.So Emil restarted with two PCR to amplify the correct GFP and to linearize the Backbone K823024(Pxyl), Emil followed the Onetaq+Phusion PCR protocol. ", "tags" : "pXyl-EFE-B0015" }