Team:Lethbridge
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University of Lethbridge IGEM team
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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:
![](https://static.igem.org/mediawiki/2010/c/c3/100505JV-EnzymeTest1Cropped.jpg)
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:
![](https://static.igem.org/mediawiki/2010/b/b0/100510JRV-EnzymeTest1Cropped.jpg)
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:
![](https://static.igem.org/mediawiki/2010/c/c3/100505JV-EnzymeTest1Cropped.jpg)
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:
![](https://static.igem.org/mediawiki/2010/b/b0/100510JRV-EnzymeTest1Cropped.jpg)
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: