Team:Groningen/Protocols

From 2009.igem.org

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{{Team:Groningen/Header}}
{{Team:Groningen/Header}}
[[Category:Team:Groningen]]
[[Category:Team:Groningen]]
-
<h1>Protocols</h1>
+
=Protocols=
-
<h2>Cloning</h2>
+
==Cloning==
-
<h3>[http://openwetware.org/wiki/PCR PCR]</h3>
+
===[http://openwetware.org/wiki/PCR PCR]===
-
<h3>Plasmid isolation</h3>
+
21 μL mastermix<sup>*</sup><BR>
-
<h3>Restriction</h3>
+
1 μL forward primer<BR>
-
<h3>Gel isolation</h3>
+
1 μL reverse primer<BR>
-
<h3>Annealing synthetic oligo’s<h3>
+
1 μL template<BR>
-
<h3>Ligation</h3>
+
1 μL Taq polymerase<BR>
-
<h3>Making competent cells</h3>
+
 
-
<h3>Transformation</h3>
+
<sup>*</sup>master mix contains:<BR>
-
<h2>Quality control</h2>
+
100 μL Taq NH<sub>4</sub><BR>
-
<h3>Colony PCR</h3>
+
8 μL dNTP's <BR>
-
<h3>Restriction analysis</h3>
+
80 μL MgCl<BR>
-
<h1>List of solutions</h1>
+
652 μL MilliQ water<BR>
-
<h2>Antibiotics</h2>
+
<BR>
-
<h3>[http://openwetware.org/wiki/Ampicillin Ampicillin]</h3>
+
<u><b>PCR Reaction</b></u>
 +
* Hotstart
 +
** 95 °C, 2 min.
 +
*25 cycles
 +
** 95 °C, 30 sec.
 +
** 61 °C, 20 sec.
 +
** 72 °C, 1.5 min.
 +
*End
 +
** 72 °C, 10 min.
 +
** 4 °C, ∞
 +
 
 +
===Plasmid isolation===
 +
===Restriction===
 +
* Cut plasmids containing promotor with <i>SpeI</i> and <i>PstI</i>
 +
** 1 μL 10x fast digest buffer
 +
** 0.5 μL <i>SpeI</i>  
 +
** 0.5 μL <i>PstI</i>
 +
** 8 μL Vector
 +
* Incubate 1 h @ 37 °C
 +
* Purify cut plasmid using PCR clean up kit
 +
===Gel isolation===
 +
* Put restriction on 1% Agarose (TBE) gel
 +
** Cut band of desired bp out
 +
** Purify using gel purification kit
 +
*** End volume 30 μL<sup>*</sup>
 +
<sup>*</sup>End volume determines concentration, variations are possible
 +
===Annealing synthetic oligo’s===
 +
====Protocol #3====
 +
<u>Restriction vectors</u>
 +
* Mix
 +
** 2 μL Tango digest buffer (Fermentas)
 +
** 16 μL vector
 +
** 1 μL [http://www.fermentas.com/catalog/re/ecori.htm <i>EcoRI</i>] (Non-fast digest)
 +
** 1 μL [http://www.fermentas.com/catalog/re/bcui.htm <i>SpeI</i>] (Non-fast digest)
 +
* Incubate 1.5 h @ 37 °C
 +
* Put to 1% agarose (1xTBE)
 +
** Isolate ~3000 bp vector
 +
** Check for presence of cut out [http://partsregistry.org/Part:BBa_P1010:Design ccdB death gene] (~600 bp)
 +
* Purify vector using gel purification kit
 +
** End volume 30 μL H<sub>2</sub>O (MilliQ)
 +
** Determine concentration using nanodrop
 +
** Store to 4 °C until ligation
 +
----
 +
<u>Phosphorylation of 5' ends & hybridization<SUP><FONT SIZE="-1">[[Silver: Oligonucleotide Inserts| [1]]]</FONT></SUP></u>
 +
*(SENSE) Mix:
 +
**3 μL 100 µM sense oligo
 +
**1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])
 +
**2 μL 10mM ATP
 +
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)]
 +
**3 μL MilliQ
 +
***(for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
 +
<BR>
 +
*(ANTI-SENSE) Mix:
 +
**3 μL 100 µM anti-sense oligo
 +
**1 μL 10 x PNK (polynucleotide kinase) buffer ([http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm Fermentas Buffer A])
 +
**2 μL 10mM ATP
 +
**1 μL [http://www.fermentas.com/catalog/modifyingenzymes/t4polynucleotidekinase.htm T4 polynucleotide kinase (PNK)]
 +
**3 μL MilliQ
 +
***(for selfcloser control, do not add oligo's. Instead 6 μL MilliQ in total)
 +
*Incubate @ 37 °C for 1.5 hours.
 +
*Mix
 +
**10 μL Sense mixture
 +
**10 μL Anti-sense mixture
 +
**3 μL 0.5 M NaCl
 +
*Place in boiling water for 3 min., and allow the reaction to cool to room temperature.
 +
----
 +
<u>Ligation</u>  
 +
*Mix
 +
**1 μL T4 ligase buffer
 +
**7.5 μL restricted vector (purified from gel)
 +
**1 μL annealing mix
 +
**0.5 μL T4 ligase
 +
*Incubate
 +
:* 1h RT
 +
:or
 +
:* ON @ 4 °C
 +
----
 +
<u>Transformation</u>
 +
*Add 5 μL of ligation mixture to 50 μL of [[TOP10 chemically competent cells]]
 +
*Heatshock, 45 sec. 42 °C
 +
** + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
 +
** Alternatively a single cut plasmid can be taken as a ligation control
 +
**Alternative - control: 1 μL [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] or [http://partsregistry.org/Part:pSB3K3 pSB3K3] carrying ccdB deathgene
 +
*Incubate 1 h @ 37 °C, 200 RPM
 +
*Plate out on LB-agar + Kanamycin (30 μg/ml for [http://partsregistry.org/Part:pSB3K3 pSB3K3]) or Ampicillin (100 μg/mL for [http://partsregistry.org/Part:pSB1AC3 pSB1AC3])
 +
**Plate out 50 μL & 200 μL of cell suspension
 +
*Grow ON @ 37 °C
 +
----
 +
<u>Checking transformations</u>
 +
*See if - control is empty for functioning antibiotics and death gene
 +
*See how many colonies on + control for functioning competent cells
 +
*See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
 +
*If enough transformants, inoculate 3 - 5 colonies in an ON culture
 +
**Alternatively perform colony PCR
 +
===Ligation===
 +
* Ligation
 +
** μL ligation buffer
 +
**1 μL ligase
 +
** μL vector
 +
** μL insert
 +
** Incubate
 +
===Making competent cells===
 +
Competent cells: TOP10 & DB3.1
 +
*10 mL ON culture is used to inoculate LB, 100 μL ON culture per 20 mL<sup>*</sup>
 +
*Cultures are grown @ 37 °C until an OD<sub>600</sub> of 0.2 ~ 0.3 is reached.
 +
*Cultures are spinned down 5 min. @ 4000 rpm, 4 °C
 +
*Supernatant is removed and pellet is resuspended in 5 mL chilled 0.1 M CaCl2
 +
**Suspension is incubated on ice for 10 min.
 +
*Suspensions are spinned down 5' @ 4000 rpm, 4 °C
 +
**Pellets were left on ice for undetermined time due to lack of liquid nitrogen
 +
*Supernatant is removed and pellet is resuspended in 2000 μL chilled 0.1 M CaCl2+10% glycerol prior to making aliquots.
 +
*Cells are divided in 50 μL aliquotes
 +
*Cells are stored @ -80 °C
 +
<sup>*</sup> Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.
 +
===Transformation===
 +
==Quality control==
 +
===Colony PCR===
 +
===Restriction analysis===
 +
=List of solutions=
 +
==Antibiotics==
 +
===[http://openwetware.org/wiki/Ampicillin Ampicillin]===
100 mg/ml Ampicillin (1000x) Stock
100 mg/ml Ampicillin (1000x) Stock
* 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
* 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
Line 22: Line 142:
* Filter sterilize 0.2 μm filter and aliquot
* Filter sterilize 0.2 μm filter and aliquot
* Store -20 °C
* Store -20 °C
-
<h3>[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]</h3>
+
===[http://openwetware.org/wiki/Chloramphenicol Chloramphenicol]===
35 mg/ml Chloramphenicol (1000x) Stock
35 mg/ml Chloramphenicol (1000x) Stock
* 0.35 g in 10 mL 100% EtOH
* 0.35 g in 10 mL 100% EtOH
* Filter sterilize 0.2 μm filter and aliquot
* Filter sterilize 0.2 μm filter and aliquot
* Store -20 °C
* Store -20 °C
-
<h3>[http://openwetware.org/wiki/Kanamycin Kanamycin]</h3>
+
===[http://openwetware.org/wiki/Kanamycin Kanamycin]===
50 mg/ml Kanamycin(1000x) Stock
50 mg/ml Kanamycin(1000x) Stock
* {{todo| g in 10 mL }}
* {{todo| g in 10 mL }}

Revision as of 11:57, 17 October 2009

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Protocols

Protocols

Cloning

PCR

21 μL mastermix*
1 μL forward primer
1 μL reverse primer
1 μL template
1 μL Taq polymerase

*master mix contains:
100 μL Taq NH4
8 μL dNTP's
80 μL MgCl
652 μL MilliQ water

PCR Reaction

  • Hotstart
    • 95 °C, 2 min.
  • 25 cycles
    • 95 °C, 30 sec.
    • 61 °C, 20 sec.
    • 72 °C, 1.5 min.
  • End
    • 72 °C, 10 min.
    • 4 °C, ∞

Plasmid isolation

Restriction

  • Cut plasmids containing promotor with SpeI and PstI
    • 1 μL 10x fast digest buffer
    • 0.5 μL SpeI
    • 0.5 μL PstI
    • 8 μL Vector
  • Incubate 1 h @ 37 °C
  • Purify cut plasmid using PCR clean up kit

Gel isolation

  • Put restriction on 1% Agarose (TBE) gel
    • Cut band of desired bp out
    • Purify using gel purification kit
      • End volume 30 μL*

*End volume determines concentration, variations are possible

Annealing synthetic oligo’s

Protocol #3

Restriction vectors

  • Mix
    • 2 μL Tango digest buffer (Fermentas)
    • 16 μL vector
    • 1 μL EcoRI (Non-fast digest)
    • 1 μL SpeI (Non-fast digest)
  • Incubate 1.5 h @ 37 °C
  • Put to 1% agarose (1xTBE)
    • Isolate ~3000 bp vector
    • Check for presence of cut out ccdB death gene (~600 bp)
  • Purify vector using gel purification kit
    • End volume 30 μL H2O (MilliQ)
    • Determine concentration using nanodrop
    • Store to 4 °C until ligation

Phosphorylation of 5' ends & hybridization [1]

  • (SENSE) Mix:


  • (ANTI-SENSE) Mix:
  • Incubate @ 37 °C for 1.5 hours.
  • Mix
    • 10 μL Sense mixture
    • 10 μL Anti-sense mixture
    • 3 μL 0.5 M NaCl
  • Place in boiling water for 3 min., and allow the reaction to cool to room temperature.

Ligation

  • Mix
    • 1 μL T4 ligase buffer
    • 7.5 μL restricted vector (purified from gel)
    • 1 μL annealing mix
    • 0.5 μL T4 ligase
  • Incubate
  • 1h RT
or
  • ON @ 4 °C

Transformation

  • Add 5 μL of ligation mixture to 50 μL of TOP10 chemically competent cells
  • Heatshock, 45 sec. 42 °C
    • + control: 1 μL pSB3K3-high or pSB1AC3-high plasmid, - control: 1 μL MilliQ
    • Alternatively a single cut plasmid can be taken as a ligation control
    • Alternative - control: 1 μL pSB1AC3 or pSB3K3 carrying ccdB deathgene
  • Incubate 1 h @ 37 °C, 200 RPM
  • Plate out on LB-agar + Kanamycin (30 μg/ml for pSB3K3) or Ampicillin (100 μg/mL for pSB1AC3)
    • Plate out 50 μL & 200 μL of cell suspension
  • Grow ON @ 37 °C

Checking transformations

  • See if - control is empty for functioning antibiotics and death gene
  • See how many colonies on + control for functioning competent cells
  • See how many selfclosers and compare to samples (>10x on sample vs. selfcloser)
  • If enough transformants, inoculate 3 - 5 colonies in an ON culture
    • Alternatively perform colony PCR

Ligation

  • Ligation
    • μL ligation buffer
    • 1 μL ligase
    • μL vector
    • μL insert
    • Incubate

Making competent cells

Competent cells: TOP10 & DB3.1

  • 10 mL ON culture is used to inoculate LB, 100 μL ON culture per 20 mL*
  • Cultures are grown @ 37 °C until an OD600 of 0.2 ~ 0.3 is reached.
  • Cultures are spinned down 5 min. @ 4000 rpm, 4 °C
  • Supernatant is removed and pellet is resuspended in 5 mL chilled 0.1 M CaCl2
    • Suspension is incubated on ice for 10 min.
  • Suspensions are spinned down 5' @ 4000 rpm, 4 °C
    • Pellets were left on ice for undetermined time due to lack of liquid nitrogen
  • Supernatant is removed and pellet is resuspended in 2000 μL chilled 0.1 M CaCl2+10% glycerol prior to making aliquots.
  • Cells are divided in 50 μL aliquotes
  • Cells are stored @ -80 °C

* Cultures should be grown in the ratio 1:5 (medium:air), so 10 mL culture in a 50 mL greiner tube.

Transformation

Quality control

Colony PCR

Restriction analysis

List of solutions

Antibiotics

Ampicillin

100 mg/ml Ampicillin (1000x) Stock

  • 1 g of Ampicillin sodium salt in 10 mL of demiwater (or 50% EtOH)
  • Add NaOH or KOH to allow the Ampicillin to dissolve
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C

Chloramphenicol

35 mg/ml Chloramphenicol (1000x) Stock

  • 0.35 g in 10 mL 100% EtOH
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C

Kanamycin

50 mg/ml Kanamycin(1000x) Stock

  • g in 10 mL
  • Filter sterilize 0.2 μm filter and aliquot
  • Store -20 °C


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