Team:Lethbridge

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University of Lethbridge IGEM team

 

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  • Lab Work
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April
May

April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available
EndonucleaseOptimal Buffer**Other Buffers
EcoRVNone2xT(100%); O,G(50-100%)
EcoRIRedO(100%);R(100%)*;2xT(100%)
BcuI/SpeITangoB(50-100%);G(50-100%)
XbaITangoB,G,2xT(50-100%)
PstIOrangeR(100%); B,G,T,2xT(50-100%)
DpnITangoB,G(100%): O,R,2xT(50-100%)
*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:
Red MMper tube (µL)Total (µL)
MilliQ H2013.7555
Red Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

Tango MMper tube (µL)Total (µL)
MilliQ H2013.7555
Tango Buffer (10x)27
pUC19 (10pg/µL)27
Total19.7569

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:
LaneSample
11kb Ladder
2Tango Control
3DpnI (Tango)
4BcuI/SpeI (Tango)
5XbaI (Tango)
6EcoRI (Red)
7PstI (Red)
8Red Control
9Empty
10Empty

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:
EnzymeBufferVolume MM(µL)Volume Enzyme(µL)
PstIRed19.75.25
XbaITango19.75.25
SpeITango19.75.25
EcoRIRed19.75.25
EcoRI/SpeIRed19.5.25+.25
XbaI/SpeITango19.5.25+.25
EcoRI/PstIRed19.5.25+.25
XbaI/PstITango19.5.25+.25

Make up Master Mixes as follows:
Red MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Red Buffer (10x)211
pDNA**211

Tango MMper tube(µL)Total*(µL)
MilliQ H2015.7586.675
Tango Buffer (10x)211
pDNA**211
*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:
LaneSampleVolume Loaded (µL)
1pSB-CEYFP/PstI10
2pSB-CEYFP/EcoRI10
3pSB-CEYFP/EcoRI/PstI10
4pSB-CEYFP/EcoRI/SpeI10
5pSB-CEYFP/XbaI/PstI10
6pSB-CEYFP/XbaI10
7pSB-CEYFP/SpeI10
8pSB-CEYFP/XbaI/SpeI10
9pSB-CEYFP/Red Master Mix Control10
10pSB-CEYFP/Tango Master Mix Control10
11pSB-CEYFP/MilliQ H20 Control10
12Ladder4

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 Mixper tube (µL)Total Volume*
MilliQ H20 Water15.7563
Red Buffer (10x)28
pDNA**28
*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

Tango Master Mixper tube (µL)Total Volume*
MilliQ H20 Water15.7594.5
Tango Buffer (10x)212
pDNA**212
*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:
LaneSampleQuantity Loaded (µL)
1MM1 Control10
2MM2 Control10
3EcoRI+SpeI(N)10
4EcoRI+SpeI(O)10
5SpeI(N)10
6SpeI(O)10
7XbaI+SpeI(N)10
8XbaI+SpeI(O)10
91kb Ladder5
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.
Construct Name (2009)Construct Location (2009)
LumazineJ4
Lumazine-dTJ5,J6
sRBS-Lumazine-dTJ7,J8
pBAD-TetRI4
pBADA5,F10
sRBSD5,E10
pSB-CEYFPE5,D6
pSB-NEYFPF5,C6
C-term TagC10
N-term TagD9,D10
pTetE4
EYFPA4
CFP CompleteD4

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:
ConstructCell in Working Plasmid Box (2010)Original Cell in Old Box
sRBS-Lumazine-dTA1J7
sRNS-Lumazine-dTA2J8
CFP CompleteB6D4
LumazineA3J4
pBADA4A5
pBADA5F10
pSB-CEYFPB5
pSB-NEYFPB4
EYFPB1A4
N-term tagB2
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:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O15.5155
Red Buffer (10X)220
EcoRI0.252.5
PstI0.252.5

Pipetting Scheme for Unrestricted reactions:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O16160
Red Buffer (10X)220

Buffer Control will be 18µL MilliQ H2O + 2µL 10x Red Buffer.
Common Protocols:
  1. Competent Cell Transformation
  2. Boiling Lysis Plasmid Preparation (Miniprep)
Competent Cell Transformation
  1. Thaw 20µL of aliquotted cells (DH5α of BL21(DE3)) on ice.
  2. 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
  3. Incubate the cells on ice for 30 minutes.
  4. Heat shock the cells in a water bath at 42oC for EXACTLY 45 seconds.
  5. Incubate the cells on ice for 1 minute.
  6. Add 250µL sterile media to the cells and incubate at 37oC for 1 hour with shaking (200RPM).
  7. Plate 100µL and 50µL on prewarmed LB agar plate containing the appropriate antibiotic.
    For ligations, plate all 250µL.
  8. Leave plate for 10-15 minutes to soak the cell suspension into the agar.
  9. Flip plate over (agar on top)
  10. Incubate the plates in the 37oC incubator overnight

Boiling Lysis Plasmid Preparation (Miniprep)
  1. 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)
  2. 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!!
  3. 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>
  4. Resuspend the cell pellet in 350µL of STET.
  5. Add 25µL of the Lysozyme solution and mix by inversion.
  6. Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.
  7. Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.
  8. 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.
  9. 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.
  10. Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500µL of ice cold ethanol.
  11. Repeat above step.
  12. Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.
  13. Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate
  14. 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.
Content 6
Content 7

 

 

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