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Team:UNICAMP-EMSE Brazil/Notebook/22 August 2011
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{{Template:UNICAMP-EMSE Brazil_Notebook}} | {{Template:UNICAMP-EMSE Brazil_Notebook}} | ||
<div style="text-align: left;"> | <div style="text-align: left;"> | ||
| - | [[Image:UNICAMP-EMSE Brazil Gel 22 08.jpg|thumb|right|300px|<font color: blue>'''Electrophoresis Gel 1, | + | [[Image:UNICAMP-EMSE Brazil Gel 22 08.jpg|thumb|right|300px|<font color: blue>'''Electrophoresis Gel 1, 22/08/2011.'''</font> <br>Gel 1:Terminator/Terminator/RBS+GFP/RBS+GFP/RBS+HlyD/RBS+HlyB/RBS+TolC/HlyA/RBS+SoxR/RBS+QseB+RBS+QseC .]] |
| - | == | + | ==22 August 2011== |
| + | ===Digestion of our synthetic genes=== | ||
'''Objective:''' Cut the band of the opened vector (terminator) or cut gene (all the rest); | '''Objective:''' Cut the band of the opened vector (terminator) or cut gene (all the rest); | ||
*'''Ladder:''' 100 bp Bio-Rad | *'''Ladder:''' 100 bp Bio-Rad | ||
| Line 9: | Line 10: | ||
<br clear="all" /> | <br clear="all" /> | ||
*'''<font color=teal>Results:'''</font> | *'''<font color=teal>Results:'''</font> | ||
| + | <font align=center> | ||
{|border="1" cellpadding="5" cellspacing="0" align="center" | {|border="1" cellpadding="5" cellspacing="0" align="center" | ||
|- | |- | ||
| Line 39: | Line 41: | ||
|2800 pb | |2800 pb | ||
|2183 pb | |2183 pb | ||
| - | |Parcial digestion | + | |Parcial digestion |
|- | |- | ||
|'''RBS+TolC''' | |'''RBS+TolC''' | ||
| Line 63: | Line 65: | ||
|2800 pb | |2800 pb | ||
|2086 pb | |2086 pb | ||
| - | |Parcial digestion | + | |Parcial digestion |
| - | |} | + | |}</font> |
| - | + | <br> | |
| + | <br> | ||
<font color=teal>'''All the genes were purified using Fermentas kit (25 uL elution) and quantified through electrophoresis:'''</font> | <font color=teal>'''All the genes were purified using Fermentas kit (25 uL elution) and quantified through electrophoresis:'''</font> | ||
| - | + | [[Image:UNICAMP-EMSE_Brazil_Gel_22_08_2.jpg|thumb|right|300px|<font color: blue>'''Second Electrophoresis Gel, 22/08/2011.'''</font> <br>Gel 2: Terminator/ Terminator / RBS+GFP/ RBS+GFP / RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA /RBS+SoxR /RBS+QseB+RBS+QseC .]] | |
*'''Ladder:''' 100 bp Bio-Rad | *'''Ladder:''' 100 bp Bio-Rad | ||
*'''Objective: ''' | *'''Objective: ''' | ||
:*Quantification (volume of ladder applied 6 uL; sample: 5 uL DNA +1 uL buffer ) | :*Quantification (volume of ladder applied 6 uL; sample: 5 uL DNA +1 uL buffer ) | ||
| - | <br> | + | <br> |
*'''Samples:''' Terminator/ Terminator / RBS+GFP/ RBS+GFP / RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA /RBS+SoxR /RBS+QseB+RBS+QseC | *'''Samples:''' Terminator/ Terminator / RBS+GFP/ RBS+GFP / RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA /RBS+SoxR /RBS+QseB+RBS+QseC | ||
| Line 80: | Line 83: | ||
<div style="color:blue">'''Nd: non digested'''<br>'''D: digested''' | <div style="color:blue">'''Nd: non digested'''<br>'''D: digested''' | ||
</div> | </div> | ||
| + | |||
| + | |||
| + | |||
| + | ===Promoter testings=== | ||
| + | [[Image:UNICAMP-EMSE_Brazil_Gel_22_08_3.png|thumb|right|300px|<font color: blue>'''Third Electrophoresis Gel, 22/08/2011.'''</font> <br>Gel 2: '''L''' = 1Kb Plus DNA Ladder Invitrogen<br>'''1''' = SoxS promoter - 100ng of the synthesized construction<br>'''2''' = flhDC promoter - 100ng of the synthesized construction<br>'''3''' and '''4''' = SoxS promoter - 2ul of the miniprep<br>'''5''' and '''6''' = Constitutive promoter - 2ul of the miniprep<br>'''7''' = flhDC promoter - 2ul of the miniprep.]] | ||
| + | *'''Objective: ''' | ||
| + | :*Check the inserted promoters in our vectors | ||
| + | |||
| + | *'''Agarose gel:''' 1.5% stained with ethidium bromide | ||
| + | |||
| + | *'''Samples: ''' | ||
| + | :*L = 1Kb Plus DNA Ladder Invitrogen | ||
| + | :*1 = SoxS promoter - 100ng of the synthesized construction | ||
| + | :*2 = flhDC promoter - 100ng of the synthesized construction | ||
| + | :*3 and 4 = SoxS promoter - 2ul of the miniprep | ||
| + | :*5 and 6 = Constitutive promoter - 2ul of the miniprep | ||
| + | :*7 = flhDC promoter - 2ul of the miniprep | ||
| + | |||
| + | *'''Results and explanation''' | ||
| + | :*Again, we did not observe any band corresponding to the excised insert. Since the vectors in which the synthesized promoters are cloned have no restriction sites for the enzymes we used (EcoRI and PstI), and given that we are observing linearized vectors after the digestions, we can conclude that there is at least something (an insert) in the vector that is being cut. So, we have two possibilities: | ||
| + | ::*(1) the amount of DNA in the excised insert is too low for visualizing in the agarose gel (which is possible in the case of very short DNA fragments); | ||
| + | ::*or (2) there is a mutation in the sequence recognized by one of the restriction enzymes, possibly inserted during the synthesis process (which is very unlikely to be occurring solely in the promoter sequences). | ||
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| + | <html> | ||
| + | <img src="https://static.igem.org/mediawiki/2011/c/cb/UNICAMP-EMSE_Brazil_Stresswars.png" width="893px"></img> | ||
| + | </html> | ||
| + | |||
| + | |||
| + | <html> | ||
| + | <head> | ||
| + | <script language='JavaScript' type='text/JavaScript'> | ||
| + | |||
| + | function makeQuote() { | ||
| + | |||
| + | Q = new Array(); | ||
| + | |||
| + | |||
| + | Q[0] = "<i>I’m Luke Skywalker, I’m here to rescue you.</i>Luke, Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[1] = "<i>Watch your mouth kid, or you’ll find yourself floating home.</i>Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[2] = "<i>Evacuate in our moment of triumph? I think you overestimate their chances.</i> Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[3] = "<i>The Force is strong with this one.</i> Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[4] = "<i> I have you now!</i> Darth Vader, Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[5] = "<i> Fear is the path to the dark side. Fear leads to anger, anger leads to hate, hate leads to suffering.</i> MsC Yoda, Star Wars Episode I: The Phantom Menace (1999)" | ||
| + | Q[6] = "<i> No blasters! No blasters!.</i> A bartender, Star Wars: Episode IV-A New Hope (1977)" | ||
| + | Q[7] = "<i> Go to the center of the gravity's pull, and find your planet you will...</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)" | ||
| + | Q[8] = "<i> Clear, your mind must be if you are to discover the real villains behind the plot.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)" | ||
| + | Q[9] = "<i> Much to learn, you still have.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)" | ||
| + | Q[10] = "<i> Meditate on this, I will.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)" | ||
| + | Q[11] = "<i> Use the Force, Luke.</i> M.Sc. Yoda, Star Wars Episode II: Attack of the Clones (2002)" | ||
| + | Q[12] = "<i> May the Force be with you.</i> Mentioned several times throughout the Star Wars saga" | ||
| + | Q[13] = "<i> I really need a Jedi Bacteria.</i> Iolanda Albuquerque, UNICAMP-EMSE team advisor" | ||
| + | Q[14] = "<i> Another splendid day in Brazil!!!</i> Marc Emery, UNICAMP-EMSE team student" | ||
| + | Q[15] = "<i> Dont' cut, let your hair volume expressing all their feelings...</i> Thibault Sabattier (UNICAMP-EMSE team student) about Nemailla's hair" | ||
| + | Q[16] = "<i> What a good life!!</i> Louise Marais (UNICAMP-EMSE team student) about living in Brazil" | ||
| + | Q[17] = "<i> Gosh!I will have nigthmares tonight! </i> Danieli Gonçalves (UNICAMP-EMSE team student) about Thibault wearing Marianna's dress" | ||
| + | Q[18] = "<i> I will revenge myself against you and Marc Emery!!!</i> Nemailla Bonturi (UNICAMP-EMSE team advisor)" | ||
| + | Q[19] = "<i> Yeah, those dresses deserve us!</i> Marc Emery (UNICAMP-EMSE team student) about Marianna's dresses" | ||
| + | Q[20] = "<i> I am really starting getting worried with you guys!!</i> Iolanda Albuquerque (UNICAMP-EMSE team advisor)" | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | index = Math.floor(Math.random() * Q.length); | ||
| + | |||
| + | document.writeln(Q[index]); | ||
| + | |||
| + | } | ||
| + | |||
| + | </script> | ||
| + | |||
| + | </head> | ||
| + | |||
| + | <style> | ||
| + | #newquote {color:gray; font-family:verdana; font-size:11px; text-align:right; padding:5px;} | ||
| + | |||
| + | #newquote i {display:block; color:black; font-family:georgia, trebuchet ms; font-size:13px;} | ||
| + | |||
| + | #newquote small {display:block; text-align:right; margin-top:10px;} | ||
| + | |||
| + | ]]></b:skin> | ||
| + | </style> | ||
| + | </html> | ||
| + | |||
| + | |||
| + | <html> | ||
| + | <head> | ||
| + | <div id='newquote'> | ||
| + | |||
| + | <script language="javascript"> | ||
| + | |||
| + | makeQuote(); | ||
| + | |||
| + | </script> | ||
| + | </div> | ||
| + | </head> | ||
| + | </html> | ||
Latest revision as of 20:56, 28 September 2011
| Home | Project | Methods | Results | Data | Team | Notebook | Human Practices | Safety | Profile | Sponsors | Wix |
Contents |
Notebook
Click on a date to see what we have done!
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Gel 1:Terminator/Terminator/RBS+GFP/RBS+GFP/RBS+HlyD/RBS+HlyB/RBS+TolC/HlyA/RBS+SoxR/RBS+QseB+RBS+QseC .
22 August 2011
Digestion of our synthetic genes
Objective: Cut the band of the opened vector (terminator) or cut gene (all the rest);
- Ladder: 100 bp Bio-Rad
- Samples:
- (all digested, forgot to apply the closed vector sample in the gel) Terminator/ Terminator/ RBS+GFP/ RBS+GFP/ RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA/ RBS+SoxR/ RBS+QseB+RBS+QseC
- Results:
| Gene | Total size (gene+vector) | Linear vector size | Gene size | Result |
|---|---|---|---|---|
| Terminator | ~3300 pb | 3300 pb | No cut | Ok, complete digestion |
| RBS+GFP | ~2800 pb | 2079 pb | 735 pb | Ok, complete digestion |
| RBS+HlyD | 4296 pb | 2800 pb | 1496 pb | Ok, complete digestion |
| RBS+HlyB | 4983 pb | 2800 pb | 2183 pb | Parcial digestion |
| RBS+TolC | 4380 pb | 2800 pb | 1580 pb | Ok, complete digestion |
| HlyA | 3027 pb | 2800 pb | 227 pb | Ok, complete digestion |
| RBS+SoxR | 3323 pb | 2800 pb | 523 pb | Ok, complete digestion |
| RBS+QseB+RBS+QseC | 4886 pb | 2800 pb | 2086 pb | Parcial digestion |
All the genes were purified using Fermentas kit (25 uL elution) and quantified through electrophoresis:
Gel 2: Terminator/ Terminator / RBS+GFP/ RBS+GFP / RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA /RBS+SoxR /RBS+QseB+RBS+QseC .
- Ladder: 100 bp Bio-Rad
- Objective:
- Quantification (volume of ladder applied 6 uL; sample: 5 uL DNA +1 uL buffer )
- Samples: Terminator/ Terminator / RBS+GFP/ RBS+GFP / RBS+HlyD/ RBS+HlyB/ RBS+TolC/ HlyA /RBS+SoxR /RBS+QseB+RBS+QseC
- HlyA did not appear in the gel!! Digest and purify again !!!
Nd: non digested
D: digested
D: digested
Promoter testings
Gel 2: L = 1Kb Plus DNA Ladder Invitrogen
1 = SoxS promoter - 100ng of the synthesized construction
2 = flhDC promoter - 100ng of the synthesized construction
3 and 4 = SoxS promoter - 2ul of the miniprep
5 and 6 = Constitutive promoter - 2ul of the miniprep
7 = flhDC promoter - 2ul of the miniprep.
- Objective:
- Check the inserted promoters in our vectors
- Agarose gel: 1.5% stained with ethidium bromide
- Samples:
- L = 1Kb Plus DNA Ladder Invitrogen
- 1 = SoxS promoter - 100ng of the synthesized construction
- 2 = flhDC promoter - 100ng of the synthesized construction
- 3 and 4 = SoxS promoter - 2ul of the miniprep
- 5 and 6 = Constitutive promoter - 2ul of the miniprep
- 7 = flhDC promoter - 2ul of the miniprep
- Results and explanation
- Again, we did not observe any band corresponding to the excised insert. Since the vectors in which the synthesized promoters are cloned have no restriction sites for the enzymes we used (EcoRI and PstI), and given that we are observing linearized vectors after the digestions, we can conclude that there is at least something (an insert) in the vector that is being cut. So, we have two possibilities:
- (1) the amount of DNA in the excised insert is too low for visualizing in the agarose gel (which is possible in the case of very short DNA fragments);
- or (2) there is a mutation in the sequence recognized by one of the restriction enzymes, possibly inserted during the synthesis process (which is very unlikely to be occurring solely in the promoter sequences).
