Team:UNITN-Trento/Notebook/Labposts/07/44

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Latest revision as of 10:42, 3 October 2013

{"date" : "2013-07-20","author" : "thomas","title" : "Digestion of AraC-pBAD + EFE and Venus PCR product","content" : "This is the part two of this experiment.Since both Venus (BBa_K537006) and AraC-pBAD + EFE were in pSB1C3 vector, I had to do a PCR on Venus using primers Prefix Fwd and Suffing Rev following this protocol. The PCR product was then confirmed by an electrophoresis analysis (Venus is 729 bp and KAPA universal Ladder was adopted).

Gel Image

The PCR product was then purified and quantified at the NanoDrop.Once I obtained Venus without his backbone, I proceeded digesting all the PRC Venus product with NgoMIV and PstI and 2-3 ug of AraC-pBAD + EFE with AgeI and PstI following the PCR product digestion protocol. The digestion products were finally purified, quantified and stored at -20°C.

Quantification Results

AraC-pBAD + EFE	33.5 ng/ul
Venus PCR product	22.8 ng/ul

","tags" : "EFE-Venus"}

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+{"date" : "2013-07-20","author" : "thomas","title" : "Digestion of AraC-pBAD + EFE and Venus PCR product","content" : "<html>This is the part two of <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-18-thomas-michele\">this experiment</a>.Since both Venus (BBa_K537006) and AraC-pBAD + EFE were in pSB1C3 vector, I had to do a PCR on Venus using primers Prefix Fwd and  Suffing Rev following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#pSB1C3-linearization-PCR\">this protocol</a>. The PCR product was then confirmed by an electrophoresis analysis (Venus is 729 bp and KAPA universal Ladder was adopted). <br/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel Image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/f/fe/Tn-2013_VenusPCR_gel.jpg\" style =\"width: 450px\"></center></html>}}<html><br/>The PCR product was then purified and quantified at the NanoDrop.Once I obtained Venus without his backbone, I proceeded digesting all the PRC Venus product with NgoMIV and PstI and 2-3 ug of AraC-pBAD + EFE with AgeI and PstI following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">the PCR product digestion protocol</a>. The digestion products were finally purified, quantified and stored at -20&deg;C.<br/> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification Results|<html><center><table class=\"tn-sp-table\"><tr><td>AraC-pBAD + EFE</td><td>33.5 ng/ul</td></tr><tr><td>Venus PCR product</td><td>22.8 ng/ul</td></tr></table></center></html>}}<html></html>","tags" : "EFE-Venus"}
-	"date" : "2013-07-22",
-	"author" : "fabio",
-	"title" : " blue and red light sensors!!",
-	"content" : "<html> let’s get ready to screen!! This time I amplified my parts by pcr them out with OneTaq polymerase!! I started from the blue promoter (number A from the firt  set). Pcr sample composition: <br>5X Onetaq standard reaction buffer  10 ul <br>10 mM dNTPs   1 ul<br>Forward primer 1ul<br>Reverse primer 1 ul<br>One Taq 0.25<br>Phusion 0.3<br>Template 0.5<br>Water up to 50 ul<br>Pcr program values: <br>Initial digestion: 94° 2min <br>Digestion: 94 30 sec<br>Annealing: 60 1 min<br>Annealing: 68° 15 sec<br>Final extention: 68° 5 min<br>(30 cycles from step 3 to step 5) <br>The screening was successful : <br> </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|gel|<html><center><img src=\"https://static.igem.org/mediawiki/2013/0/06/Tn-2013_C.jpg\" width=\"450px\" /></center></html>}}<html>Finally I purified the PCR and abtained a 209,8 ng/ul yieald.Meanwhile I started thinking about the ligation of the two blue parts: the idea is to use k592016 as plasmid and k592006 as an insert to put downstream . So I had to digest o/n  3 ug of 016(from the first set)  with Spel and Pstl, and 006( the purified Pcr)  with Xbal and Pstl. At the same time I digested R0010 with Ecorl and Spel, cause I’ll need to put it on top of everything once I have my biobrick.</html>",
-	"tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"
-}