Team:Lethbridge
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<br><b><font size=+2>May 12/2010</font>(in the lab: JV)</b><br> | <br><b><font size=+2>May 12/2010</font>(in the lab: JV)</b><br> | ||
- | <b>Objective: Miniprep of plasmid DNA from transformed cells</b><br> | + | <b>Objective: Miniprep of plasmid DNA from transformed cells</b>(JV, AV, HB)<br> |
<b>Method:</b><br> | <b>Method:</b><br> | ||
<ul><li>Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).</li> | <ul><li>Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).</li> | ||
<li>Allow cells in liquid culture to grow overnight in 37<sup>o</sup>C shaking incubator (300RPM) | <li>Allow cells in liquid culture to grow overnight in 37<sup>o</sup>C shaking incubator (300RPM) | ||
Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.</li> | Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.</li> | ||
- | <li>CHANGE: Step 14, used MilliQ H<sub>2</sub>O (with 20ng/µL RNase A) instead of TE buffer.</li></ul> | + | <li>CHANGE: Step 14, used MilliQ H<sub>2</sub>O (with 20ng/µL RNase A) instead of TE buffer.</li></ul><br> |
+ | Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20<sup>o</sup>C freezer in the iGEM lab. Plasmids were placed in the following cells: | ||
+ | <table><table border="3"> | ||
+ | <tr><td>Construct</td><td>Cell in Working Plasmid Box (2010)</td><td>Original Cell in Old Box</td></tr> | ||
+ | <tr><td>sRBS-Lumazine-dT</td><td>A1</td><td>J7</td></tr> | ||
+ | <tr><td>sRNS-Lumazine-dT</td><td>A2</td><td>J8</td></tr> | ||
+ | <tr><td>CFP Complete</td><td>B6</td><td>D4</td></tr> | ||
+ | <tr><td>Lumazine</td><td>A3</td><td>J4</td></tr> | ||
+ | <tr><td>pBAD</td><td>A4</td><td>A5</td></tr> | ||
+ | <tr><td>pBAD</td><td>A5</td><td>F10</td></tr> | ||
+ | <tr><td>pSB-CEYFP</td><td>B5</td><td></td></tr> | ||
+ | <tr><td>pSB-NEYFP</td><td>B4</td><td></td></tr> | ||
+ | <tr><td>EYFP</td><td>B1</td><td>A4</td></tr> | ||
+ | <tr><td>N-term tag</td><td>B2</td><td></td></tr> | ||
+ | </table> | ||
+ | |||
+ | <b>Objective:</b> Restrict plasmid DNA with restriction endonucleases (JV)<br> | ||
+ | <b>Method:</b><br> | ||
+ | Have: | ||
+ | 10 lanes of restricted plasmid DNA <br> | ||
+ | 10 lanes of unrestricted plasmid DNA <br> | ||
+ | 1 lane of buffer control <br> | ||
+ | Use EcoRI (prefix cutter) and PstI (suffix cutter) <br><br> | ||
+ | Pipetting Scheme for Restriction Tubes: | ||
+ | <table><table border="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sup>O</td><td>15.5</td><td>155</td></tr> | ||
+ | <tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr> | ||
+ | <tr><td>EcoRI</td><td>0.25</td><td>2.5</td></tr> | ||
+ | <tr><td>PstI</td><td>0.25</td><td>2.5</td></tr> | ||
+ | </table><br> | ||
+ | Pipetting Scheme for Unrestricted reactions: | ||
+ | <table><table border="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume/tube (µL)</td><td>Total Volume*</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>160</td></tr> | ||
+ | <tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr> | ||
+ | </table><br> | ||
+ | Buffer Control will be 18µL MilliQ H<sub>2</sub>O + 2µL 10x Red Buffer.<br> | ||
Line 308: | Line 345: | ||
<div class="TabbedPanelsContent"> | <div class="TabbedPanelsContent"> | ||
<b><font size=+2>Common Protocols:</b></font><br> | <b><font size=+2>Common Protocols:</b></font><br> | ||
- | <ol><li><a href=#transformation>Competent Cell Transformation</a></li><br> | + | <ol><li><a href=#transformation>Competent Cell Transformation</a></li> |
+ | <li><a href=#miniprep>Boiling Lysis Plasmid Preparation (Miniprep)<br> | ||
</ol> | </ol> | ||
<a name="transformation"></a> | <a name="transformation"></a> | ||
Line 327: | Line 365: | ||
<li>Flip plate over (agar on top)</li> | <li>Flip plate over (agar on top)</li> | ||
<li>Incubate the plates in the 37<sup>o</sup>C incubator overnight</li> | <li>Incubate the plates in the 37<sup>o</sup>C incubator overnight</li> | ||
- | </ol> | + | </ol><br> |
- | + | <a name="miniprep"></a> | |
+ | <b>Boiling Lysis Plasmid Preparation (Miniprep)</b><br> | ||
+ | <ol> | ||
+ | <li>Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)</li> | ||
+ | <li>Remove and discard as much of the supernatant as possible by aspiration (e.g with a Pasteur Pipette). Do not suck up the cell pellet!!</li> | ||
+ | <li>Rinse the cell pellet by washing 1.0mL of sterile MilliQ H<sub>2</sub>O gently down the inside wall of the MCT. This removes any traces of the supernatant adhering to the MCT wall while minimizing the disturbance to the cell pellet. (/li> | ||
+ | <li>Resuspend the cell pellet in 350µL of STET. </li> | ||
+ | <li>Add 25µL of the Lysozyme solution and mix by inversion.</li> | ||
+ | <li>Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.</li> | ||
+ | <li>Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.</li> | ||
+ | <li>Precipitate the plasmid DNA by adding 40µL of 3.0M sodium acetate (pH 5.2) and 420µL isopropanol. Mix by inversion. Mix by inversion and incubate for 5 minutes at room temperature.</li> | ||
+ | <li>Pellet the plasmid DNA by centrifugation at 13000RPM for 10 minutes at 4<sup>o</sup>C. A pellet of plasmid DNA should be visible at the base of the MCT when complete.</li> | ||
+ | <li>Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500µL of ice cold ethanol.</li> | ||
+ | <li>Repeat above step.</li> | ||
+ | <li>Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.</li> | ||
+ | <li>Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate</li> | ||
+ | <li>Add 50µL of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20<sup>o</sup>C.</ol> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 20:11, 15 May 2010
University of Lethbridge IGEM team
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Jump to Month:
April
May
April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
*Star Activity
**Optimal Buffer from Fermentas
Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme>
Methods: Set up Master Mixes:
To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:
Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining
May 5/2010(in the lab: JV)
Objective: Test Restriction Endonucleases for activity (take 2)
Relevant Information:
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks
Prefix Enzymes are: EcoRI and XbaI
Suffix Enyzmes are: SpeI and PstI
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)
Reactions will be assembled as follows:
Make up Master Mixes as follows:
*Volume per reaction multiplied by 5.5
**Unknown concentration of pDNA
Incubated for 70min at 37oC (Start-1:05pm; End-2:15pm)
Added 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)
Added 1µL of 6x loading dye to 2µL of gene ruler 1kb ladder
Load order as follows:
Ran gel at 100V for 1 hour
Results:
This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes
Conclusion: Test other source of SpeI to see if it has any activity.
May 6/2010(in the lab:KG, AS)
Objective: To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.
Method:
*Volume per tube multiplied by 4
**Used pSB NEYFP pDNA from cell E5 in plasmid box
Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix
*Volume per tube multiplied by 6
**Used pSB NEYFP pDNA from cell E5 in plasmid box
Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix
Incubated all reactions at 37oC for 1h (Start-8:30pm; End-9:30pm)
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning
Tube Names:
Master Mix 1 Control (Red Buffer)
Master Mix 2 Control (Tango Buffer)
E+S(N); EcoRI + SpeI(N)
E+S(O); EcoRI + SpeI(O)
X+S(N); XbaI + SpeI(N)
X+S(O); XbaI + SpeI(O)
S(N); SpeI(N)
S(O); SpeI(O)
Placed in -20oC freezer of later analysis by agarose electrophoresis
May 10/2010(in the lab:JV)
Objective: To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis
Method:
Run gel for 60min at 100V
Results:
It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.
May 10/2010(in the lab:JV, KG, AV)
Objective:Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.
Method:Make 2L of LB media with agar
2x10g Tryptone
2X2.5g Yeast Extract
2x5g NaCl
2x10g Agar
Continued May 11/2010
(Stock Ampicillin solution is 100mg/mL)
Have 4x500mL of LB with Agar
Add 500µL of stock ampicillin to 500mL of media
May 11/2010 Evening (in the lab: KG, AV, MC, TF, JV, JS)
Objective: To transform the following plasmids into DH5α E.coli cells.
Method: Followed "Competent Cell Transformation" protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.
Results: The following plasmids were successfully transformed and formed colonies:
Conclusion: Need another attempt to transform the following plasmids:
May 12/2010(in the lab: JV)
Objective: Miniprep of plasmid DNA from transformed cells(JV, AV, HB)
Method:
Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20oC freezer in the iGEM lab. Plasmids were placed in the following cells:
April
May
April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
Endonuclease | Optimal Buffer** | Other Buffers |
EcoRV | None | 2xT(100%); O,G(50-100%) |
EcoRI | Red | O(100%);R(100%)*;2xT(100%) |
BcuI/SpeI | Tango | B(50-100%);G(50-100%) |
XbaI | Tango | B,G,2xT(50-100%) |
PstI | Orange | R(100%); B,G,T,2xT(50-100%) |
DpnI | Tango | B,G(100%): O,R,2xT(50-100%) |
**Optimal Buffer from Fermentas
Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme>
Methods: Set up Master Mixes:
Red MM | per tube (µL) | Total (µL) |
MilliQ H20 | 13.75 | 55 |
Red Buffer (10x) | 2 | 7 |
pUC19 (10pg/µL) | 2 | 7 |
Total | 19.75 | 69 |
Tango MM | per tube (µL) | Total (µL) |
MilliQ H20 | 13.75 | 55 |
Tango Buffer (10x) | 2 | 7 |
pUC19 (10pg/µL) | 2 | 7 |
Total | 19.75 | 69 |
To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:
Lane | Sample |
1 | 1kb Ladder |
2 | Tango Control |
3 | DpnI (Tango) |
4 | BcuI/SpeI (Tango) |
5 | XbaI (Tango) |
6 | EcoRI (Red) |
7 | PstI (Red) |
8 | Red Control |
9 | Empty |
10 | Empty |
Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining
May 5/2010(in the lab: JV)
Objective: Test Restriction Endonucleases for activity (take 2)
Relevant Information:
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks
Prefix Enzymes are: EcoRI and XbaI
Suffix Enyzmes are: SpeI and PstI
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)
Reactions will be assembled as follows:
Enzyme | Buffer | Volume MM(µL) | Volume Enzyme(µL) |
PstI | Red | 19.75 | .25 |
XbaI | Tango | 19.75 | .25 |
SpeI | Tango | 19.75 | .25 |
EcoRI | Red | 19.75 | .25 |
EcoRI/SpeI | Red | 19.5 | .25+.25 |
XbaI/SpeI | Tango | 19.5 | .25+.25 |
EcoRI/PstI | Red | 19.5 | .25+.25 |
XbaI/PstI | Tango | 19.5 | .25+.25 |
Make up Master Mixes as follows:
Red MM | per tube(µL) | Total*(µL) |
MilliQ H20 | 15.75 | 86.675 |
Red Buffer (10x) | 2 | 11 |
pDNA** | 2 | 11 |
Tango MM | per tube(µL) | Total*(µL) |
MilliQ H20 | 15.75 | 86.675 |
Tango Buffer (10x) | 2 | 11 |
pDNA** | 2 | 11 |
**Unknown concentration of pDNA
Incubated for 70min at 37oC (Start-1:05pm; End-2:15pm)
Added 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)
Added 1µL of 6x loading dye to 2µL of gene ruler 1kb ladder
Load order as follows:
Lane | Sample | Volume Loaded (µL) |
1 | pSB-CEYFP/PstI | 10 |
2 | pSB-CEYFP/EcoRI | 10 |
3 | pSB-CEYFP/EcoRI/PstI | 10 |
4 | pSB-CEYFP/EcoRI/SpeI | 10 |
5 | pSB-CEYFP/XbaI/PstI | 10 |
6 | pSB-CEYFP/XbaI | 10 |
7 | pSB-CEYFP/SpeI | 10 |
8 | pSB-CEYFP/XbaI/SpeI | 10 |
9 | pSB-CEYFP/Red Master Mix Control | 10 |
10 | pSB-CEYFP/Tango Master Mix Control | 10 |
11 | pSB-CEYFP/MilliQ H20 Control | 10 |
12 | Ladder | 4 |
Ran gel at 100V for 1 hour
Results:
This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes
Conclusion: Test other source of SpeI to see if it has any activity.
May 6/2010(in the lab:KG, AS)
Objective: To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.
Method:
Red Master Mix | per tube (µL) | Total Volume* |
MilliQ H20 Water | 15.75 | 63 |
Red Buffer (10x) | 2 | 8 |
pDNA** | 2 | 8 |
**Used pSB NEYFP pDNA from cell E5 in plasmid box
Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix
Tango Master Mix | per tube (µL) | Total Volume* |
MilliQ H20 Water | 15.75 | 94.5 |
Tango Buffer (10x) | 2 | 12 |
pDNA** | 2 | 12 |
**Used pSB NEYFP pDNA from cell E5 in plasmid box
Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix
Incubated all reactions at 37oC for 1h (Start-8:30pm; End-9:30pm)
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning
Tube Names:
Master Mix 1 Control (Red Buffer)
Master Mix 2 Control (Tango Buffer)
E+S(N); EcoRI + SpeI(N)
E+S(O); EcoRI + SpeI(O)
X+S(N); XbaI + SpeI(N)
X+S(O); XbaI + SpeI(O)
S(N); SpeI(N)
S(O); SpeI(O)
Placed in -20oC freezer of later analysis by agarose electrophoresis
May 10/2010(in the lab:JV)
Objective: To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis
Method:
Lane | Sample | Quantity Loaded (µL) |
1 | MM1 Control | 10 |
2 | MM2 Control | 10 |
3 | EcoRI+SpeI(N) | 10 |
4 | EcoRI+SpeI(O) | 10 |
5 | SpeI(N) | 10 |
6 | SpeI(O) | 10 |
7 | XbaI+SpeI(N) | 10 |
8 | XbaI+SpeI(O) | 10 |
9 | 1kb Ladder | 5 |
Results:
It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.
May 10/2010(in the lab:JV, KG, AV)
Objective:Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.
Method:Make 2L of LB media with agar
2x10g Tryptone
2X2.5g Yeast Extract
2x5g NaCl
2x10g Agar
Continued May 11/2010
(Stock Ampicillin solution is 100mg/mL)
Have 4x500mL of LB with Agar
Add 500µL of stock ampicillin to 500mL of media
May 11/2010 Evening (in the lab: KG, AV, MC, TF, JV, JS)
Objective: To transform the following plasmids into DH5α E.coli cells.
Construct Name (2009) | Construct Location (2009) |
Lumazine | J4 |
Lumazine-dT | J5,J6 |
sRBS-Lumazine-dT | J7,J8 |
pBAD-TetR | I4 |
pBAD | A5,F10 |
sRBS | D5,E10 |
pSB-CEYFP | E5,D6 |
pSB-NEYFP | F5,C6 |
C-term Tag | C10 |
N-term Tag | D9,D10 |
pTet | E4 |
EYFP | A4 |
CFP Complete | D4 |
Method: Followed "Competent Cell Transformation" protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.
Results: The following plasmids were successfully transformed and formed colonies:
- Lumazine (J4)
- sRBS-Lumazine-dT (J7)
- sRBS-Lumazine-dT (J8)
- pBAD (A5)
- pBAD (F10)
- pSB-CEYFP
- pSB-NEYFP
- N-term tag
- EYFP (A4)
- CFP Complete (D4)
Conclusion: Need another attempt to transform the following plasmids:
- Lumazine-dT (J5,J6)
- pBAD-TetR
- sRBS (D5,E10)
- C-Term tag
- pTet
May 12/2010(in the lab: JV)
Objective: Miniprep of plasmid DNA from transformed cells(JV, AV, HB)
Method:
- Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).
- Allow cells in liquid culture to grow overnight in 37oC shaking incubator (300RPM) Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.
- CHANGE: Step 14, used MilliQ H2O (with 20ng/µL RNase A) instead of TE buffer.
Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20oC freezer in the iGEM lab. Plasmids were placed in the following cells: