Team:Lethbridge

From 2010.igem.org

(Difference between revisions)
Line 345: Line 345:
<tr><td>EcoRI</td><td>0.25</td><td>2.5</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>
<tr><td>PstI</td><td>0.25</td><td>2.5</td></tr>
-
</table><br>
+
</table>
 +
*Amount per tube multiplied by 10<br>
Pipetting Scheme for Unrestricted reactions:
Pipetting Scheme for Unrestricted reactions:
<table><table border="3">
<table><table border="3">
Line 351: Line 352:
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>160</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>
<tr><td>Red Buffer (10X)</td><td>2</td><td>20</td></tr>
-
</table><br>
+
</table>
 +
*Amount per tube multiplied by 10<br>
Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
Place in 37<sup>o</sup>C water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.<br>
Place in 37<sup>o</sup>C water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.<br>
Line 380: Line 382:
<tr><td>19</td><td>sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
<tr><td>19</td><td>sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
<tr><td>20</td><td>Restricted sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
<tr><td>20</td><td>Restricted sRBS-Lumazine-dT (J8)</td><td>5</td></tr>
-
</table>
+
</table><br>
-
 
+
Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)<br>
-
<b>Results:</b><br><br>
+
Stained with ethidium bromide for 20 minutes.<br>
 +
<b>Results:</b><br>
 +
<a href="https://static.igem.org/mediawiki/2010/7/79/100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg"><img src="https://static.igem.org/mediawiki/2010/7/79/100513TF.KG.JS-Plasmids_for_Seq.Cropped.jpg"  width="225" height="151"/></a><br><br>
<b><font size=+2>May 13/2010 Evening</font>(in lab: AS,TF,KG,JS,MC)</b><br>
<b><font size=+2>May 13/2010 Evening</font>(in lab: AS,TF,KG,JS,MC)</b><br>
<b>Objective:</b> To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:<br>
<b>Objective:</b> To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:<br>
Line 393: Line 397:
<li>pTet</li></ul><br>
<li>pTet</li></ul><br>
All DH5&alpha; cells were used up in the last transformation, had to aliquot an additional 50x 20&micro;L aliquots (MC,TF)<br>
All DH5&alpha; cells were used up in the last transformation, had to aliquot an additional 50x 20&micro;L aliquots (MC,TF)<br>
-
Transform plasmid DNA into newly aliquotted DH5&alpha; cells. (KG,JS)<br>
+
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5&alpha; cells. (KG,JS)<br>
-
NOTE: 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).<br>
+
NOTES:<br>
 +
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).<br>
 +
Plated all 250&micro;L of culture.<br>
 +
<b>Results:</b><br>
 +
<table><table border="3">
 +
<tr><td><b>Construct</td><td>Result</b></td></tr>
 +
<tr><td>Lumazine-dT(1)</td><td>Growth present</td></tr>
 +
<tr><td>sRBS-Lumazine-dT</td><td>Growth present</td></tr>
 +
<tr><td>sRBS (D5)</td><td>Growth present</td></tr>
 +
<tr><td>sRBS (E10)</td><td>Growth present</td></tr>
 +
<tr><td>C-term tag</td><td>No growth present</td></tr>
 +
<tr><td>pTet</td><td>No growth present</td></tr>
 +
</table><br>
 +
<b>Next Steps:</b><br>
 +
Make another attempt to transform the C-term tag and pTet constructs.<br>
 +
Start overnight cultures of cells that grew for plasmid prep and sequencing.<br><br>
 +
 
 +
<b><font size=+2>May 14/2010</font>(in the lab: JV)</b><br>
 +
<b>Objective:</b> Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.<br>
 +
<b>Method:</b> Measure absorbance of samples at 260nm.<br>
 +
<b>Results:</b><br>
 +
<table><table border="3">
 +
<tr><td><b>Sample</td><td>Absorbance at 260nm</b></td></tr>
 +
<tr><td>sRBS-Lumazine-dT (J7)</td><td>0.311</td></tr>
 +
<tr><td>sRBS-Lumazine-dT (J8)</td><td>0.309</td></tr>
 +
<tr><td>CFP complete</td><td>0.316</td></tr>
 +
<tr><td>N-term tag</td><td>0.290</td></tr>
 +
<tr><td>pSB-CEYFP</td><td>0.338</td></tr>
 +
<tr><td>pSB-NEYFP</td><td>0.403</td></tr>
 +
<tr><td>pBAD (A5)</td><td>0.282</td></tr>
 +
<tr><td>pBAD (F10)</td><td>0.562</td></tr>
 +
<tr><td>EYFP</td><td>0.389</td></tr>
 +
<tr><td>Lumazine</td><td>0.221</td></tr>
 +
</table><br>
 +
<b>Conclusion:</b> All plasmids present in sufficient concentrations for sequence analysis.<br>
 +
 
 +
<b>Objective:</b> Purify plasmid DNA from cells recently transformed.<br>
 +
<b>Method:</b>
 +
<ul>
 +
<li>Inoculate 5mL of sterile LB liquid media (with 100&micro;g/mL ampicillin) with cells picked from colonies of transformation plates, including the following:<br>
 +
Lumazine-dT (J5)<br>
 +
pBad-TetR<br>
 +
sRBS (D5,E10)<br></li>
 +
<li>NOTE: Lumazine-dT did NOT grow overnight</li>
 +
<li>Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. <b>(May 15/2010; JV,TF)</b><br>
 +
NOTE: Added 50&micro;L of MilliQ H<sub>2</sub>O (with RNase A at a concentration of 20ng/&micro;L) to dissolve pDNA instead of TE buffer.<br><br></li></ul>
 +
<b>Objective:</b> Perform restriction digest on the above prepared plasmid DNA.<br>
 +
<b>Method:</b><br>
 +
Used EcoRI as prefix cutter and PstI as suffix cutter.<br>
 +
Pipetting Scheme for Restriction Tubes:
 +
<table><table border="3">
 +
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
 +
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>56</td></tr>
 +
<tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr>
 +
<tr><td>EcoRI</td><td>0.25</td><td>0.875</td></tr>
 +
<tr><td>PstI</td><td>0.25</td><td>0.875</td></tr>
 +
</table>
 +
*Amount per tube multiplied by 3.5<br>
 +
Add 18&micro;L master mix to each plasmid DNA sample<br>
 +
Pipetting Scheme for Unrestricted reactions:
 +
<table><table border="3">
 +
<tr><td><b>Ingredient</b></td><td>Volume/tube (&micro;L)</td><td>Total Volume*</td></tr>
 +
<tr><td>MilliQ H<sub>2</sup>O</td><td>16</td><td>56</td></tr>
 +
<tr><td>Red Buffer (10X)</td><td>2</td><td>7</td></tr>
 +
</table>
 +
*Amount per tube multiplied by 3.5<br>
 +
Add 18&micro;L master mix to each plasmid DNA sample<br>
 +
Buffer Control will be 18&micro;L MilliQ H<sub>2</sub>O + 2&micro;L 10x Red Buffer.<br>
 +
Place in 37<sup>o</sup>C water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.<br>

Revision as of 23:32, 15 May 2010

Untitled Document

University of Lethbridge IGEM team

 

  • Main
  • Team
  • Projects
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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

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

Also generated sterile glycerol stocks and placed in -80oC freezer in the 2010 iGEM box as follows:
ConstructCell Working Glycerol Stock Box (2010)
sRBS-Lumazine-dT (J7)B2,C4,D2
sRNS-Lumazine-dT (J8)C6
CFP CompleteA10, C8
LumazineA8,B10
pBAD (from A5)B5,B9
pBAD (from F10)B3,B7
pSB-CEYFPC3,B5
pSB-NEYFPB6,C1
EYFPC7,B8
N-term tagC2,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:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O15.5155
Red Buffer (10X)220
EcoRI0.252.5
PstI0.252.5
*Amount per tube multiplied by 10
Pipetting Scheme for Unrestricted reactions:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O16160
Red Buffer (10X)220
*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:
LaneSampleVolume Loaded (µL)
11 kb Ladder5
2Buffer Control5
3pSB-NEYFP5
4Restricted Lumazine5
5Lumazine5
6Restricted pSB-NEYFP5
7pSB-CEYFP5
8Restricted pSB-CEYFP5
9pBAD5
10Restricted pBAD5
11EYFP5
12Restricted EYFP5
13CFP Complete5
14Restricted CFP Complete5
15sRBS-Lumazine-dT (J7)5
16Restricted sRBS-Lumazine-dT (J7)5
17N-term Tag5
18Restricted N-term Tag5
19sRBS-Lumazine-dT (J8)5
20Restricted 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:
ConstructResult
Lumazine-dT(1)Growth present
sRBS-Lumazine-dTGrowth present
sRBS (D5)Growth present
sRBS (E10)Growth present
C-term tagNo growth present
pTetNo 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:
SampleAbsorbance at 260nm
sRBS-Lumazine-dT (J7)0.311
sRBS-Lumazine-dT (J8)0.309
CFP complete0.316
N-term tag0.290
pSB-CEYFP0.338
pSB-NEYFP0.403
pBAD (A5)0.282
pBAD (F10)0.562
EYFP0.389
Lumazine0.221

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.

Objective: Perform restriction digest on the above prepared plasmid DNA.
Method:
Used EcoRI as prefix cutter and PstI as suffix cutter.
Pipetting Scheme for Restriction Tubes:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O1656
Red Buffer (10X)27
EcoRI0.250.875
PstI0.250.875
*Amount per tube multiplied by 3.5
Add 18µL master mix to each plasmid DNA sample
Pipetting Scheme for Unrestricted reactions:
IngredientVolume/tube (µL)Total Volume*
MilliQ H2O1656
Red Buffer (10X)27
*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.
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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