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Team:UNICAMP-EMSE Brazil/Notebook/23 August 2011
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===Transformation=== | ===Transformation=== | ||
The plasmids purified yesterday were transformed using the [https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/protocols/chimiocompetent_prepare Chemically Competent ''E. coli''] and the protocol of [https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/protocols/chimiocompetent_prepare Chemo-transformation]. | The plasmids purified yesterday were transformed using the [https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/protocols/chimiocompetent_prepare Chemically Competent ''E. coli''] and the protocol of [https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/protocols/chimiocompetent_prepare Chemo-transformation]. | ||
| - | *Three plates for each Construct were | + | *Three plates for each Construct were used, inoculated with different volumes of the transformation: |
:*100 uL, 200 uL and the remaining volume after centrifugation and concentration of bacteria for the plasmid containing: | :*100 uL, 200 uL and the remaining volume after centrifugation and concentration of bacteria for the plasmid containing: | ||
::*RBS+QseB+QseC+Terminator | ::*RBS+QseB+QseC+Terminator | ||
| Line 58: | Line 58: | ||
::*Both photos are from the same gel, but the right one is in grey scale. The single digestions (conducted with only one enzyme - lanes 1 and 2) showed that we have both restriction sites in the vector, which is actually present in our synthesized insert because the vector does not have any restriction site. So, there is no mutations here. Lane three shows our released promoter (black arrow), although it is not easy to see. | ::*Both photos are from the same gel, but the right one is in grey scale. The single digestions (conducted with only one enzyme - lanes 1 and 2) showed that we have both restriction sites in the vector, which is actually present in our synthesized insert because the vector does not have any restriction site. So, there is no mutations here. Lane three shows our released promoter (black arrow), although it is not easy to see. | ||
::*So, our promoters are probably correctly inserted in the vectors, with the problem being that they are too short to be visualized after staining with ethidium bromide. Since we do not need to take them off the vector to work with them (we can put our larger fragments in the vector with the promoters), as soon as we have the gene(s)+terminator constructions done we can start putting them in the linearized vectors. | ::*So, our promoters are probably correctly inserted in the vectors, with the problem being that they are too short to be visualized after staining with ethidium bromide. Since we do not need to take them off the vector to work with them (we can put our larger fragments in the vector with the promoters), as soon as we have the gene(s)+terminator constructions done we can start putting them in the linearized vectors. | ||
| + | |||
| + | |||
| + | |||
| + | ===Digestions recipe=== | ||
| + | *'''HlyA:''' | ||
| + | :*50ul - HlyA miniprep | ||
| + | :*8ul - milli-Q water | ||
| + | :*7ul - 10X Buffer R | ||
| + | :*1ul - EcoRI | ||
| + | :*4ul - SpeI | ||
| + | :*'''Total = 70ul''' | ||
| + | |||
| + | |||
| + | *'''RBS+HlyD:''' | ||
| + | :*32ul - RBS+HlyD miniprep | ||
| + | :*4ul - 10X Buffer R | ||
| + | :*0.8ul - EcoRI | ||
| + | :*3.2ul - SpeI | ||
| + | :*'''Total = 40ul''' | ||
| + | |||
| + | ===Ligations recipe=== | ||
| + | *'''RBS+TolC - vector+terminator''' | ||
| + | :*9ul milli-Q water | ||
| + | :*5ul - RBS+TolC (~300ng) | ||
| + | :*3ul - vector+terminator (~100ng) | ||
| + | :*2ul - 10X T4 Buffer | ||
| + | :*1ul - T4 DNA Ligase | ||
| + | :*'''Total = 20ul''' | ||
| + | |||
| + | *'''vector+terminator - RBS+SoxR''' | ||
| + | :*3ul - milli-Q water | ||
| + | :*20ul - RBS+SoxR (~80ng) | ||
| + | :*2.5ul - vector+terminator (~80ng) | ||
| + | :*3ul - 10X T4 Buffer | ||
| + | :*1.5ul - T4 DNA Ligase | ||
| + | :*'''TOTAL = 30ul''' | ||
| + | |||
| + | *'''RBS+QseB+RBS+QseC - vector+terminator''' | ||
| + | :*4ul - milli-Q water | ||
| + | :*10ul - RBS+QseB+RBS+QseC (~200ng) | ||
| + | :*3ul - vector+terminator (~100ng) | ||
| + | :*2ul - 10X T4 Buffer | ||
| + | :*1ul - T4 DNA Ligase | ||
| + | :*TOTAL = 20ul | ||
| + | |||
| + | Incubate all ligation reactions for 1hour at 22°C. | ||
| + | Use 1-2ul of these ligations to do the transformation in the afternoon. | ||
| + | |||
| + | |||
| + | '''Next Tasks:''' | ||
| + | #Incubate both reactions at 37°C for 3-4hours. | ||
| + | #Electrophoresis (loading the whole digestions volume) | ||
| + | #Purify the bands | ||
| + | #Electrophoresis to confirm | ||
| + | <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 00:07, 29 September 2011
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Contents |
Notebook
Click on a date to see what we have done!
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Gel 1:: Ladder / HlyD digested (three joint wells) / HlyD non-digested / HlyA digested (three joint wells) / HlyA non-digested .
23 August 2011
Purification of digested HlyA and RBS+HlyD - secretion system
Objective: Electrophoresis for the purification of digested HlyA and RBS+HlyD.
- Ladder: Bio-Rad 100bp – 10.000bp
- Agarose concentration: 1%
- Samples:
- Ladder / HlyD digested (three joint wells) / HlyD non-digested / HlyA digested (three joint wells) / HlyA non-digested
- Results:
| Gene | Total size (gene+vector) | Linear vector size | Gene size | Result |
|---|---|---|---|---|
| HlyA | 3027 pb | 2800 pb | 227 pb | Size OK & complete digestion |
| RBS+HlyD | 4296 pb | 2800 pb | 1496 pb | Size OK & complete digestion |
-All the genes were purified using Fermentas kit.
Transformation
The plasmids purified yesterday were transformed using the Chemically Competent E. coli and the protocol of Chemo-transformation.
- Three plates for each Construct were used, inoculated with different volumes of the transformation:
- 100 uL, 200 uL and the remaining volume after centrifugation and concentration of bacteria for the plasmid containing:
- RBS+QseB+QseC+Terminator
- RBS+TolC+Terminator
- RBS+SoxR+Terminator
New digestion for our synthetic promoters and Electrophoresis in a more concentrated gel
Objective: Check for problems with our synthesized promoters
- Agarose concentration: 1.5% stained with ethidium bromide
- Samples:
- L = 1Kb Plus DNA Ladder (Invitrogen)
- 1 = vector containing the SoxS promoter digested with EcoRI
- 2 = vector containing the SoxS promoter digested with PstI
- 3 = vector containing the SoxS promoter digested with EcoRI and PstI
- Explanation of results:
- Both photos are from the same gel, but the right one is in grey scale. The single digestions (conducted with only one enzyme - lanes 1 and 2) showed that we have both restriction sites in the vector, which is actually present in our synthesized insert because the vector does not have any restriction site. So, there is no mutations here. Lane three shows our released promoter (black arrow), although it is not easy to see.
- So, our promoters are probably correctly inserted in the vectors, with the problem being that they are too short to be visualized after staining with ethidium bromide. Since we do not need to take them off the vector to work with them (we can put our larger fragments in the vector with the promoters), as soon as we have the gene(s)+terminator constructions done we can start putting them in the linearized vectors.
Digestions recipe
- HlyA:
- 50ul - HlyA miniprep
- 8ul - milli-Q water
- 7ul - 10X Buffer R
- 1ul - EcoRI
- 4ul - SpeI
- Total = 70ul
- RBS+HlyD:
- 32ul - RBS+HlyD miniprep
- 4ul - 10X Buffer R
- 0.8ul - EcoRI
- 3.2ul - SpeI
- Total = 40ul
Ligations recipe
- RBS+TolC - vector+terminator
- 9ul milli-Q water
- 5ul - RBS+TolC (~300ng)
- 3ul - vector+terminator (~100ng)
- 2ul - 10X T4 Buffer
- 1ul - T4 DNA Ligase
- Total = 20ul
- vector+terminator - RBS+SoxR
- 3ul - milli-Q water
- 20ul - RBS+SoxR (~80ng)
- 2.5ul - vector+terminator (~80ng)
- 3ul - 10X T4 Buffer
- 1.5ul - T4 DNA Ligase
- TOTAL = 30ul
- RBS+QseB+RBS+QseC - vector+terminator
- 4ul - milli-Q water
- 10ul - RBS+QseB+RBS+QseC (~200ng)
- 3ul - vector+terminator (~100ng)
- 2ul - 10X T4 Buffer
- 1ul - T4 DNA Ligase
- TOTAL = 20ul
Incubate all ligation reactions for 1hour at 22°C. Use 1-2ul of these ligations to do the transformation in the afternoon.
Next Tasks:
- Incubate both reactions at 37°C for 3-4hours.
- Electrophoresis (loading the whole digestions volume)
- Purify the bands
- Electrophoresis to confirm
