Team:UNITN-Trento/Notebook/Labposts/07/49

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(Difference between revisions)

Latest revision as of 10:43, 3 October 2013

{"date" : "2013-07-22","author" : "fabio","title" : " blue and red light sensors!!","content" : " 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:
5X Onetaq standard reaction buffer 10 ul
10 mM dNTPs 1 ul
Forward primer 1ul
Reverse primer 1 ul
One Taq 0.25
Phusion 0.3
Template 0.5
Water up to 50 ul
Pcr program values:
Initial digestion: 94° 2min
Digestion: 94 30 sec
Annealing: 60 1 min
Annealing: 68° 15 sec
Final extention: 68° 5 min
(30 cycles from step 3 to step 5)
The screening was successful :

gel

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.","tags" : " YF1_FixJ - FixK2- ho1_PcyA_cph8-OmpR"}

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+{"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"}
-	"date" : "2013-07-23",
-	"author" : "caterina",
-	"title" : "Trasformation of SAM",
-	"content" : "<html>I performed the ligation and the trasformation of SAMsynthetase in pSB1C3 with ROO10.<br/><br/>Also I transformed Gabriele's ligations from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">17/07</a> into NEB10&beta;.</html>",
-	"tags" : "R0010-SAMsynthetase"
-}