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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:
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:
Also generated sterile glycerol stocks and placed in -80oC freezer in the 2010 iGEM box as follows:
Objective: Restrict plasmid DNA with restriction endonucleases (JV)
Method:
Have: 10 lanes of restricted plasmid DNA
10 lanes of unrestricted plasmid DNA
1 lane of buffer control
Use EcoRI (prefix cutter) and PstI (suffix cutter)
Pipetting Scheme for Restriction Tubes:
*Amount per tube multiplied by 10
Pipetting Scheme for Unrestricted reactions:
*Amount per tube multiplied by 10
Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.
Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 2µL of 6x DNA loading dye to 6µL of TAE buffer and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:
Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)
Stained with ethidium bromide for 20 minutes.
Results:
May 13/2010 Evening(in lab: AS,TF,KG,JS,MC)
Objective: To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:
All DH5α cells were used up in the last transformation, had to aliquot an additional 50x 20µL aliquots (MC,TF)
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5α cells. (KG,JS)
NOTES:
AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).
Plated all 250µL of culture.
Results:
Next Steps:
Make another attempt to transform the C-term tag and pTet constructs.
Start overnight cultures of cells that grew for plasmid prep and sequencing.
May 14/2010(in the lab: JV)
Objective: Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.
Method: Measure absorbance of samples at 260nm.
Results:
Conclusion: All plasmids present in sufficient concentrations for sequence analysis.
Objective: Purify plasmid DNA from cells recently transformed.
Method:
Method:
Used EcoRI as prefix cutter and PstI as suffix cutter.
Pipetting Scheme for Restriction Tubes:
*Amount per tube multiplied by 3.5
Add 18µL master mix to each plasmid DNA sample
Pipetting Scheme for Unrestricted reactions:
*Amount per tube multiplied by 3.5
Add 18µL master mix to each plasmid DNA sample
Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.
Analyze samples on a 1% agarose gel (small gel apparatus).
Add 3.3µL of 6x DNA loading dye to each reaction mixture and load.
Ran gel at 100V for 75 minutes (Start-2:30pm; End-3:45pm)
Stained in ethidium bromide for 10 minutes
Results:
Picture to come.....
There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).
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:
| Construct | Cell in Working Plasmid Box (2010) | Original Cell in Old Box |
| sRBS-Lumazine-dT | A1 | J7 |
| sRNS-Lumazine-dT | A2 | J8 |
| CFP Complete | B6 | D4 |
| Lumazine | A3 | J4 |
| pBAD | A4 | A5 |
| pBAD | A5 | F10 |
| pSB-CEYFP | B5 | |
| pSB-NEYFP | B4 | |
| EYFP | B1 | A4 |
| N-term tag | B2 |
Also generated sterile glycerol stocks and placed in -80oC freezer in the 2010 iGEM box as follows:
| Construct | Cell Working Glycerol Stock Box (2010) |
| sRBS-Lumazine-dT (J7) | B2,C4,D2 |
| sRNS-Lumazine-dT (J8) | C6 |
| CFP Complete | A10, C8 |
| Lumazine | A8,B10 |
| pBAD (from A5) | B5,B9 |
| pBAD (from F10) | B3,B7 |
| pSB-CEYFP | C3,B5 |
| pSB-NEYFP | B6,C1 |
| EYFP | C7,B8 |
| N-term tag | C2,D4 |
Objective: Restrict plasmid DNA with restriction endonucleases (JV)
Method:
Have: 10 lanes of restricted plasmid DNA
10 lanes of unrestricted plasmid DNA
1 lane of buffer control
Use EcoRI (prefix cutter) and PstI (suffix cutter)
Pipetting Scheme for Restriction Tubes:
| Ingredient | Volume/tube (µL) | Total Volume* |
| MilliQ H2O | 15.5 | 155 |
| Red Buffer (10X) | 2 | 20 |
| EcoRI | 0.25 | 2.5 |
| PstI | 0.25 | 2.5 |
Pipetting Scheme for Unrestricted reactions:
| Ingredient | Volume/tube (µL) | Total Volume* |
| MilliQ H2O | 16 | 160 |
| Red Buffer (10X) | 2 | 20 |
Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.
Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 2µL of 6x DNA loading dye to 6µL of TAE buffer and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:
| Lane | Sample | Volume Loaded (µL) |
| 1 | 1 kb Ladder | 5 |
| 2 | Buffer Control | 5 |
| 3 | pSB-NEYFP | 5 |
| 4 | Restricted Lumazine | 5 |
| 5 | Lumazine | 5 |
| 6 | Restricted pSB-NEYFP | 5 |
| 7 | pSB-CEYFP | 5 |
| 8 | Restricted pSB-CEYFP | 5 |
| 9 | pBAD | 5 |
| 10 | Restricted pBAD | 5 |
| 11 | EYFP | 5 |
| 12 | Restricted EYFP | 5 |
| 13 | CFP Complete | 5 |
| 14 | Restricted CFP Complete | 5 |
| 15 | sRBS-Lumazine-dT (J7) | 5 |
| 16 | Restricted sRBS-Lumazine-dT (J7) | 5 |
| 17 | N-term Tag | 5 |
| 18 | Restricted N-term Tag | 5 |
| 19 | sRBS-Lumazine-dT (J8) | 5 |
| 20 | Restricted sRBS-Lumazine-dT (J8) | 5 |
Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)
Stained with ethidium bromide for 20 minutes.
Results:
May 13/2010 Evening(in lab: AS,TF,KG,JS,MC)
Objective: To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:
- Lumazine-dT (J5,J6)
- pBad-TetR
- sRBS (D5,E10)
- C-term tag
- pTet
All DH5α cells were used up in the last transformation, had to aliquot an additional 50x 20µL aliquots (MC,TF)
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5α cells. (KG,JS)
NOTES:
AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).
Plated all 250µL of culture.
Results:
| Construct | Result |
| Lumazine-dT(1) | Growth present |
| sRBS-Lumazine-dT | Growth present |
| sRBS (D5) | Growth present |
| sRBS (E10) | Growth present |
| C-term tag | No growth present |
| pTet | No growth present |
Next Steps:
Make another attempt to transform the C-term tag and pTet constructs.
Start overnight cultures of cells that grew for plasmid prep and sequencing.
May 14/2010(in the lab: JV)
Objective: Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.
Method: Measure absorbance of samples at 260nm.
Results:
| Sample | Absorbance at 260nm |
| sRBS-Lumazine-dT (J7) | 0.311 |
| sRBS-Lumazine-dT (J8) | 0.309 |
| CFP complete | 0.316 |
| N-term tag | 0.290 |
| pSB-CEYFP | 0.338 |
| pSB-NEYFP | 0.403 |
| pBAD (A5) | 0.282 |
| pBAD (F10) | 0.562 |
| EYFP | 0.389 |
| Lumazine | 0.221 |
Conclusion: All plasmids present in sufficient concentrations for sequence analysis.
Objective: Purify plasmid DNA from cells recently transformed.
Method:
- Inoculate 5mL of sterile LB liquid media (with 100µg/mL ampicillin) with cells picked from colonies of transformation plates, including the following:
Lumazine-dT (J5)
pBad-TetR
sRBS (D5,E10) - NOTE: Lumazine-dT did NOT grow overnight
- Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. (May 15/2010; JV,TF)
NOTE: Added 50µL of MilliQ H2O (with RNase A at a concentration of 20ng/µL) to dissolve pDNA instead of TE buffer.
Method:
Used EcoRI as prefix cutter and PstI as suffix cutter.
Pipetting Scheme for Restriction Tubes:
| Ingredient | Volume/tube (µL) | Total Volume* |
| MilliQ H2O | 16 | 56 |
| Red Buffer (10X) | 2 | 7 |
| EcoRI | 0.25 | 0.875 |
| PstI | 0.25 | 0.875 |
Add 18µL master mix to each plasmid DNA sample
Pipetting Scheme for Unrestricted reactions:
| Ingredient | Volume/tube (µL) | Total Volume* |
| MilliQ H2O | 16 | 56 |
| Red Buffer (10X) | 2 | 7 |
Add 18µL master mix to each plasmid DNA sample
Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Place in 37oC water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.
Analyze samples on a 1% agarose gel (small gel apparatus).
Add 3.3µL of 6x DNA loading dye to each reaction mixture and load.
| Lane | Sample | Volume Loaded (µL) |
| 1 | 1 kb Ladder | 4 |
| 2 | Restricted sRBS (E10) | 10 |
| 3 | sRBS (E10) | 10 |
| 4 | Restricted sRBS (D5) | 10 |
| 5 | sRBS (D5) | 10 |
| 6 | Restricted sRBS-Lumazine-dT | 10 |
| 7 | sRBS-Lumazine-dT | 10 |
| 8 | Red Buffer Control | 10 |
Stained in ethidium bromide for 10 minutes
Results:
Picture to come.....
There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).
Common Protocols:
Competent Cell Transformation
Boiling Lysis Plasmid Preparation (Miniprep)
Competent Cell Transformation
- Thaw 20µL of aliquotted cells (DH5α of BL21(DE3)) on ice.
- Gently pipet 2.0µL of DNA into competent cells
ATTENTION:
Do not perform any additional mixing
Never use more DNA that 10% of the volume of the competent cells otherwise the cells get destroyed by osmotic shock - Incubate the cells on ice for 30 minutes.
- Heat shock the cells in a water bath at 42oC for EXACTLY 45 seconds.
- Incubate the cells on ice for 1 minute.
- Add 250µL sterile media to the cells and incubate at 37oC for 1 hour with shaking (200RPM).
- Plate 100µL and 50µL on prewarmed LB agar plate containing the appropriate antibiotic.
For ligations, plate all 250µL. - Leave plate for 10-15 minutes to soak the cell suspension into the agar.
- Flip plate over (agar on top)
- Incubate the plates in the 37oC incubator overnight
Boiling Lysis Plasmid Preparation (Miniprep)
- 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)
- 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!!
- Rinse the cell pellet by washing 1.0mL of sterile MilliQ H2O 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>
- Resuspend the cell pellet in 350µL of STET.
- Add 25µL of the Lysozyme solution and mix by inversion.
- Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.
- Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.
- 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.
- Pellet the plasmid DNA by centrifugation at 13000RPM for 10 minutes at 4oC. A pellet of plasmid DNA should be visible at the base of the MCT when complete.
- Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500µL of ice cold ethanol.
- Repeat above step.
- Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.
- Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate
- 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 -20oC.
Updated May 19/2010
Row A
Row B
Row C
Row D
Row E
Row F
Row G
Row H
Row I
Row J
Row A
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| A1 | |||
| A2 | |||
| A3 | |||
| A4 | |||
| A5 | |||
| A6 | |||
| A7 | |||
| A8 | |||
| A9 | |||
| A10 |
Row B
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| B1 | |||
| B2 | |||
| B3 | |||
| B4 | |||
| B5 | |||
| B6 | |||
| B7 | |||
| B8 | |||
| B9 | |||
| B10 |
Row C
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| C1 | |||
| C2 | |||
| C3 | |||
| C4 | |||
| C5 | |||
| C6 | |||
| C7 | |||
| C8 | |||
| C9 | |||
| C10 |
Row D
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| D1 | |||
| D2 | |||
| D3 | |||
| D4 | |||
| D5 | |||
| D6 | |||
| D7 | |||
| D8 | |||
| D9 | |||
| D10 |
Row E
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| E1 | |||
| E2 | |||
| E3 | |||
| E4 | |||
| E5 | |||
| E6 | |||
| E7 | |||
| E8 | |||
| E9 | |||
| E10 |
Row F
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| F1 | |||
| F2 | |||
| F3 | |||
| F4 | |||
| F5 | |||
| F6 | |||
| F7 | |||
| F8 | |||
| F9 | |||
| F10 |
Row G
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| G1 | |||
| G2 | |||
| G3 | |||
| G4 | |||
| G5 | |||
| G6 | |||
| G7 | |||
| G8 | |||
| G9 | |||
| G10 |
Row H
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| H1 | |||
| H2 | |||
| H3 | |||
| H4 | |||
| H5 | |||
| H6 | |||
| H7 | |||
| H8 | |||
| H9 | |||
| H10 |
Row I
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| I1 | |||
| I2 | |||
| I3 | |||
| I4 | |||
| I5 | |||
| I6 | |||
| I7 | |||
| I8 | |||
| I9 | |||
| I10 |
Row J
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| J1 | |||
| J2 | |||
| J3 | |||
| J4 | |||
| J5 | |||
| J6 | |||
| J7 | |||
| J8 | |||
| J9 | |||
| J10 |
Updated May 19/2010
Row A
Row B
Row C
Row D
Row E
Row F
Row G
Row H
Row I
Row J
Row A
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| A1 | |||
| A2 | |||
| A3 | |||
| A4 | |||
| A5 | |||
| A6 | |||
| A7 | |||
| A8 | |||
| A9 | |||
| A10 |
Row B
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| B1 | |||
| B2 | |||
| B3 | |||
| B4 | |||
| B5 | |||
| B6 | |||
| B7 | |||
| B8 | |||
| B9 | |||
| B10 |
Row C
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| C1 | |||
| C2 | |||
| C3 | |||
| C4 | |||
| C5 | |||
| C6 | |||
| C7 | |||
| C8 | |||
| C9 | |||
| C10 |
Row D
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| D1 | |||
| D2 | |||
| D3 | |||
| D4 | |||
| D5 | |||
| D6 | |||
| D7 | |||
| D8 | |||
| D9 | |||
| D10 |
Row E
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| E1 | |||
| E2 | |||
| E3 | |||
| E4 | |||
| E5 | |||
| E6 | |||
| E7 | |||
| E8 | |||
| E9 | |||
| E10 |
Row F
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| F1 | |||
| F2 | |||
| F3 | |||
| F4 | |||
| F5 | |||
| F6 | |||
| F7 | |||
| F8 | |||
| F9 | |||
| F10 |
Row G
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| G1 | |||
| G2 | |||
| G3 | |||
| G4 | |||
| G5 | |||
| G6 | |||
| G7 | |||
| G8 | |||
| G9 | |||
| G10 |
Row H
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| H1 | |||
| H2 | |||
| H3 | |||
| H4 | |||
| H5 | |||
| H6 | |||
| H7 | |||
| H8 | |||
| H9 | |||
| H10 |
Row I
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| I1 | |||
| I2 | |||
| I3 | |||
| I4 | |||
| I5 | |||
| I6 | |||
| I7 | |||
| I8 | |||
| I9 | |||
| I10 |
Row J
| Cell | Common Name | Standard Registry of Parts ID | Date Entered |
| J1 | |||
| J2 | |||
| J3 | |||
| J4 | |||
| J5 | |||
| J6 | |||
| J7 | |||
| J8 | |||
| J9 | |||
| J10 |
Content 6
Content 7
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