Team:Lethbridge

From 2010.igem.org

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<b><font size=+2>Common Protocols:</b></font><br>
<b><font size=+2>Common Protocols:</b></font><br>
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<a href=#transformation>Competent Cell Transformation</a>
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<ol><li><a href=#transformation>Competent Cell Transformation</a></li><br>
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<a name="transformation"></a>
<b>Competent Cell Transformation</b><br>
<b>Competent Cell Transformation</b><br>
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ATTENTION: <br>
ATTENTION: <br>
Do not perform any additional mixing<br>
Do not perform any additional mixing<br>
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Never use more DNA that <u>10% of the volume of the competent cells<u> otherwise the cells get destroyed by osmotic shock</li>
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Never use more DNA that <u>10% of the volume of the competent cells<u> otherwise the cells</u> get destroyed by osmotic shock</li>
<li>Incubate the cells on ice for 30 minutes.</li>
<li>Incubate the cells on ice for 30 minutes.</li>
<li>Heat shock the cells in a water bath at <u>42<sup>o</sup>C for EXACTLY 45 seconds.</u></li>
<li>Heat shock the cells in a water bath at <u>42<sup>o</sup>C for EXACTLY 45 seconds.</u></li>

Revision as of 02:32, 15 May 2010

Untitled Document

University of Lethbridge IGEM team

 

  • Main
  • Team
  • Projects
  • Notebook
  • Meetings
  • Parts
  • Collaborations
blah balh balh

Our team is based yada yada

  • Team Pics
  • Tab 3
  • Team bios
Team pictures
Content 3
Pics of us

 

 

 

 

 

Overview

  • Project 1
  • Porejct 2
Content 1
Content 2

 

 

 

 

  • Lab Work
  • Common Protocols
Jump to Month:
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

May 13
Common Protocols:
  1. Competent Cell Transformation

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
Content 6
Content 7

 

 

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