Team:Queens-Canada/protocols

From 2010.igem.org

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h1. Protocols
+
{{:Team:Queens-Canada/head}}
-
====Rehydration====
+
<h1>Miscellaneous Protocols</h1>
-
Latest revision: *June 3, 2010*
+
You can open and collapse protocols by clicking on their titles.
-
======Materials======
+
<html><div class="section"><h2>Rehydration</h2></html>
 +
 
 +
Latest revision: '''June 3, 2010'''
 +
 
 +
<h3>Materials</h3>
* kit plate (from iGEM parts distribution)
* kit plate (from iGEM parts distribution)
-
* diH ~2~ O
+
* diH<sub>2</sub>O
-
======Procedure======
+
<h3>Procedure</h3>
-
# Using sterile technique, load *10μL* of diH ~2~ O into a p20 pipette.
+
# Using sterile technique, load '''10 μL''' of diH<sub>2</sub>O into a p20 pipette.
# Pierce the foil of the correct well on the distribution plate.
# Pierce the foil of the correct well on the distribution plate.
# Firmly but carefully lower the pipette tip into the very bottom of the well.
# Firmly but carefully lower the pipette tip into the very bottom of the well.
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# Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.
# Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.
# Write "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.
# Write "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.
-
# Ensuring that you keep the plate level, put it into a *4°C* fridge, and leave it there for one hour to allow all of the DNA to dissolve.
+
# Ensuring that you keep the plate level, put it into a '''4° C''' fridge, and leave it there for one hour to allow all of the DNA to dissolve.
# Transfer the entire DNA solution from the well into an Eppendorf tube.
# Transfer the entire DNA solution from the well into an Eppendorf tube.
-
====Heat Shock Transformation====
+
<html></div></html>
 +
 
 +
<html><div class="section"><h2>Heat Shock</h2></html>
-
Latest revision: *June 3, 2010*
+
Latest revision: '''June 3, 2010'''
-
======Materials======
+
<h3>Materials</h3>
-
* *100μL* of chemical competent cells (TOP10) per transformation  
+
*''' 100 μL''' of chemical competent cells (TOP10) per transformation  
* Miniprep plasmid DNA
* Miniprep plasmid DNA
* 2XTY or SOC medium  
* 2XTY or SOC medium  
* Antibiotic plates (according to plasmid)
* Antibiotic plates (according to plasmid)
-
======Procedure======
+
<h3>Procedure</h3>
-
# Take out competent cells from -*80°C* and put them on ice immediately before they are needed.  
+
# Take out competent cells from -'''80° C''' and put them on ice immediately before they are needed.  
-
# Add *2μl* plasmid DNA to thawed cells and mix by flicking the side of the tube. Incubate on ice for *20* minutes.  
+
# Add '''2 μL''' plasmid DNA to thawed cells and mix by flicking the side of the tube. Incubate on ice for *20''' minutes.  
-
# Pre-warm antibiotic plates in *37°C* incubator.  
+
# Pre-warm antibiotic plates in '''37° C''' incubator.  
-
# Heat shock for *1* min *15* sec at *42°C*.
+
# Heat shock for '''1''' min '''15''' sec at '''42° C'''.
-
# Place on Ice for *2* minutes.  
+
# Place on Ice for '''2''' minutes.  
-
# Add *500μl* 2XTY (or SOC) medium (kept at room temp.) to each tube.  
+
# Add '''500 μL''' 2XTY (or SOC) medium (kept at room temp.) to each tube.  
-
# Shake the tubes at *37°C* for *1* hour on a shaking incubator.  
+
# Shake the tubes at '''37° C''' for '''1''' hour on a shaking incubator.  
-
# Spread *100μl* of each transformation tube on appropriate antibiotic plates.   
+
# Spread '''100 μL''' of each transformation tube on appropriate antibiotic plates.   
-
# Incubate at *37°C* overnight.
+
# Incubate at '''37° C''' overnight.
-
====Liquid Culture====
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<html></div></html>
-
Latest revision: *June 3, 2010*
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<html><div class="section"><h2>Liquid Culture</h2></html>
-
======Materials======
+
Latest revision: '''June 3, 2010'''
 +
 
 +
<h3>Materials</h3>
* Glass or plastic culture tubes  
* Glass or plastic culture tubes  
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* Parafilm
* Parafilm
-
======Procedure======
+
<h3>Procedure</h3>
# Flame a glass pipette, open the bottle of medium and flame the mouth.
# Flame a glass pipette, open the bottle of medium and flame the mouth.
-
# Withdraw amount you need to fill your tubes (*5ml* per tube), flame the cap and recap the bottle as quickly as possible.
+
# Withdraw amount you need to fill your tubes ('''5 mL''' per tube), flame the cap and recap the bottle as quickly as possible.
-
# Remove the tube cap, flame the top of the culture tube, pipette in *5ml*, flame the top of the tube and cap it.
+
# Remove the tube cap, flame the top of the culture tube, pipette in '''5 mL''', flame the top of the tube and cap it.
# Pick up one colony using a P20 pipette tip. Uncap the tube, flame the top, inject the tip into the tube.
# Pick up one colony using a P20 pipette tip. Uncap the tube, flame the top, inject the tip into the tube.
-
# Incubate the tubes at *37°C* overnight or until cells have reached the desired concentration. This should take between *12* and *16* hours.
+
# Incubate the tubes at '''37° C''' overnight or until cells have reached the desired concentration. This should take between '''12''' and '''16''' hours.
-
# Seal the transformed bacteria culture plate(s) that were used with Parafilm and store in *4°C* fridge.
+
# Seal the transformed bacteria culture plate(s) that were used with Parafilm and store in '''4° C''' fridge.
-
====Glycerol Stock====
+
<html></div></html>
-
Latest revision: *June 3, 2010*
+
<html><div class="section"><h2>Glycerol Stock</h2></html>
-
======Materials======
+
Latest revision: '''June 3, 2010'''
 +
 
 +
<h3>Materials</h3>
* Overnight bacterial cell culture
* Overnight bacterial cell culture
* Cryogenic screw-cap vials
* Cryogenic screw-cap vials
-
* 30% glycerol in H ~2~ O
+
* 30% glycerol in H<sub>2</sub>O
-
======Procedure======
+
<h3>Procedure</h3>
-
# Pipette *750μl* 30% glycerol into cryogenic vials. (Note: withdraw very slowly as glycerol is very viscous)  
+
# Pipette '''750 μL''' 30% glycerol into cryogenic vials. (Note: withdraw very slowly as glycerol is very viscous)  
-
# Add *750μl* of overnight culture to each vial.  
+
# Add '''750 μL''' of overnight culture to each vial.  
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.  
# Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.  
-
# Label each vial in accordance with labeling convention.
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# Label each vial in accordance with labelling convention.
-
# Store in a freeze box in a -*80⁰C* freezer.
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# Store in a freeze box in a -'''80° C''' freezer.
-
====Miniprep====
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<html></div></html>
-
Latest revision: *June 3, 2010*
+
<html><div class="section"><h2>Miniprep</h2></html>
-
======Materials======
+
Latest revision: '''June 3, 2010'''
-
Refer to miniprep kit
+
<h3>Materials</h3>
-
======Procedure======
+
''Refer to miniprep kit''
-
Refer to miniprep kit
+
<h3>Procedure</h3>
-
====Digestion====
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''Refer to miniprep kit''
-
Latest revision: *June 3, 2010*
+
<html></div></html>
-
======Materials======
+
<html><div class="section"><h2>Digestion</h2></html>
 +
 
 +
Latest revision: '''June 3, 2010'''
 +
 
 +
<h3>Materials</h3>
* Digestion Buffer
* Digestion Buffer
* Enzyme(s)
* Enzyme(s)
* DNA
* DNA
-
* ddH ~2~ O
+
* ddH<sub>2</sub>O
-
======Procedure======
+
<h3>Procedure</h3>
-
# Remove the digestion buffer and enzymes from the -*20C* freezer, and place on ice
+
# Remove the digestion buffer and enzymes from the -'''20° C''' freezer, and place on ice
-
# Add *14µL* of water to an Eppendorf tube
+
# Add '''14 µL''' of water to an Eppendorf tube
# Thaw the digestion buffer and the enzymes
# Thaw the digestion buffer and the enzymes
# Add digestion buffer to tube
# Add digestion buffer to tube
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# Incubate (time and temperature are variable and depend upon the enzymes used)
# Incubate (time and temperature are variable and depend upon the enzymes used)
-
====Ligation====
+
<html></div></html>
-
Latest revision: *June 3, 2010*
+
<html><div class="section"><h2>Ligation</h2></html>
-
======Materials======
+
Latest revision: '''June 3, 2010'''
-
|Linear vector DNA|*20-100* ng|
+
<h3>Materials</h3>
-
|Insert DNA|*6:1* molar ratio of insert to vector|
+
<div class="aside" style="width: auto; display: inline-block">
-
|*5X* T4 ligase buffer|*4µl*|
+
{|
-
|T4 DNA Ligase|*1µl*|
+
|Linear vector DNA
-
|Water, nuclease-free|*15µl* – vector and insert vol |
+
|'''20-100''' ng
-
|Total volume|*20µl*|
+
|-
 +
|Insert DNA
 +
|'''6:1''' molar ratio of insert to vector
 +
|-
 +
|'''5X''' T4 ligase buffer
 +
|'''4 µL'''
 +
|-
 +
|T4 DNA Ligase
 +
|'''1 µL'''
 +
|-
 +
|Water, nuclease-free
 +
|'''15 µL''' – vector and insert vol  
 +
|-
 +
|Total volume
 +
|'''20 µL'''
 +
|-
 +
|}</div>
-
======Procedure======
+
<h3>Procedure</h3>
# Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase
# Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase
-
# Transfer *5 µl* of the mastermix to each properly labeled sample tube.  
+
# Transfer '''5 µL''' of the mastermix to each properly labeled sample tube.  
-
# Add corresponding insert DNA and vector DNA into the tubes. Top the volume to *20* µl using ddH ~2~ O.  
+
# Add corresponding insert DNA and vector DNA into the tubes. Top the volume to '''20''' µl using ddH<sub>2</sub>O.  
-
# Incubate one hour at *22⁰C* (or room temperature)  
+
# Incubate one hour at '''22° C''' (or room temperature)  
-
# Heat inactivate T4 DNA ligase at *65°C* for *10* min.  
+
# Heat inactivate T4 DNA ligase at '''65° C''' for '''10''' min.  
-
# Use up to *5 µl* of the mixture for transformation of chemically competent cells.
+
# Use up to '''5 µL''' of the mixture for transformation of chemically competent cells.
 +
 
 +
<html></div></html>
-
====Gel Electrophoresis====
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<html><div class="section"><h2>Gel Electrophoresis</h2></html>
-
Latest revision: *June 3, 2010*
+
Latest revision: '''June 3, 2010'''
-
======Materials======
+
<h3>Materials</h3>
* Loading dye
* Loading dye
* 100kb DNA ladder  
* 100kb DNA ladder  
-
* 1% agarose gel (*50ml* 1X TBE and *1g* agarose)  
+
* ''' 1% agarose gel ('''50 mL''' 1X TBE and '''1 g''' agarose)  
* Gel box and power supply
* Gel box and power supply
* Ethidium bromide stain  
* Ethidium bromide stain  
* UV box/gel imager
* UV box/gel imager
-
======Procedure======
+
<h3>Procedure</h3>
-
======Making Agarose Gel======
+
<h3>Making Agarose Gel</h3>
-
Weigh out *0.5g* of agarose and add to a *250ml* Erlenmeyer flask.  
+
Weigh out '''0.5 g''' of agarose and add to a '''250 mL''' Erlenmeyer flask.  
-
# Add *50ml* 1X TBE to the flask and mix by swirling.   
+
# Add '''50 mL''' 1X TBE to the flask and mix by swirling.   
-
# Microwave TBE agarose until the solution becomes clear and obtains a uniform consistency. (First microwave for *1min* and then for *30sec* intervals. DO NOT allow the solution to boil over in the microwave).  
+
# Microwave TBE agarose until the solution becomes clear and obtains a uniform consistency. (First microwave for '''1 min''' and then for '''30 sec''' intervals. DO NOT allow the solution to boil over in the microwave).  
-
# Use glove to remove flask from the microwave. Allow flask to cool on the lab bench for *5 min* (But do not wait until the gel starts to polymerize)  
+
# Use glove to remove flask from the microwave. Allow flask to cool on the lab bench for '''5 min''' (But do not wait until the gel starts to polymerize)  
-
# Take out EtBr from -*20⁰C* freezer. Add *3μl* to *50ml* TBE agarose and swirl the flask to mix. Return EtBr to the freezer immediately after use. (Note: EtBr is a carcinogen and a mutagen. Always use glove and lab coat, if available, to handle things contaminated with EtBr.)
+
# Take out EtBr from -'''20° C''' freezer. Add '''3 μL''' to '''50 mL''' TBE agarose and swirl the flask to mix. Return EtBr to the freezer immediately after use. (Note: EtBr is a carcinogen and a mutagen. Always use glove and lab coat, if available, to handle things contaminated with EtBr.)
# Carefully pour TBE agarose into the casting tray to avoid bubbles. Make sure the tray is placed on a flat surface. Insert comb into the TAE agarose gel.  
# Carefully pour TBE agarose into the casting tray to avoid bubbles. Make sure the tray is placed on a flat surface. Insert comb into the TAE agarose gel.  
-
# Let the gel polymerize for *20min.*
+
# Let the gel polymerize for '''20 min'''.
-
======Sample Preparation======
+
<h3>Sample Preparation</h3>
-
# Add appropriate amount of loading dye such that the mixture of loading dye and sample contains *1X* concentration of dye. If using *5X* dye, use 4 parts sample and 1 part dye.
+
# Add appropriate amount of loading dye such that the mixture of loading dye and sample contains '''1X''' concentration of dye. If using '''5X''' dye, use 4 parts sample and 1 part dye.
# Use appropriate volume of ladder (depends on the ladder used).
# Use appropriate volume of ladder (depends on the ladder used).
-
======Electrophoresis======
+
<h3>Electrophoresis</h3>
# Once the gel is solidified, remove the comb carefully and place the casting tray in the gel box. Make sure the wells point towards the black (negative) electrode.  
# Once the gel is solidified, remove the comb carefully and place the casting tray in the gel box. Make sure the wells point towards the black (negative) electrode.  
# Fill the gel box with TBE until the entire gel is immersed in solution.  
# Fill the gel box with TBE until the entire gel is immersed in solution.  
# Load prepared samples into the wells. Slowly pull out the pipette tip from the well before releasing the piston of the pipette. This avoids inserting bubbles into the wells, which will disturb the sample.  
# Load prepared samples into the wells. Slowly pull out the pipette tip from the well before releasing the piston of the pipette. This avoids inserting bubbles into the wells, which will disturb the sample.  
-
# Close the lid of the gel box. Run the gel at *100V* constant voltage for *1* hour.
+
# Close the lid of the gel box. Run the gel at '''100 V''' constant voltage for '''1''' hour.
-
======Imaging======
+
<h3>Imaging</h3>
# Turn off the gel box power supply.  
# Turn off the gel box power supply.  
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# Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door.
# Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door.
-
====PCR====
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<html></div></html>
-
Latest revision: *June 3, 2010*
+
<html><div class="section"><h2>PCR</h2></html>
-
======Materials======
+
Latest revision: '''June 3, 2010'''
 +
<h3>Materials</h3>
 +
 +
<div class="aside" style="width: auto; display: inline-block">
{|
{|
-
|*10X* PCR buffer (w/ MgCl2)
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|'''10X''' PCR buffer (w/ MgCl<sub>2</sub>)
-
|*5.0* μl
+
|'''5.0''' μL
|-
|-
-
|*2* mM dNTP mix
+
|'''25''' mM dNTP mix
-
|*5.0* μl
+
|'''5.0''' μL
|-
|-
-
|*10μM* ddH2O
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|'''10μM''' ddH2O
-
|*33.5* μl
+
|'''33.5''' μL
|-
|-
-
|Forward primer (*10μM)
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|Forward primer ('''10μM''')
-
|*2.0* μl
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|'''2.0''' μL
|-
|-
-
|Reverse primer (*10μM)
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|Reverse primer ('''10μM''')
-
|*2.0* μl
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|'''2.0''' μL
|-
|-
|Template DNA
|Template DNA
-
|*2.0* μl
+
|'''2.0''' μL
|-
|-
|DNA polymerase (eg. Taq)
|DNA polymerase (eg. Taq)
-
|*0.5* μl (*1.25unit)
+
|'''0.5''' μL ('''1.25 unit''')
|-
|-
|Total volume
|Total volume
-
|*50* μl
+
|'''50''' μL
|}
|}
 +
</div>
-
======Procedure======
+
<h3>Procedure</h3>
# Label PCR tubes with sample names. Always include a negative control sample, which lacks template DNA.  
# Label PCR tubes with sample names. Always include a negative control sample, which lacks template DNA.  
-
# Take out reagents from -*20⁰C* freezer and put them on ice at all times, especially DNA polymerase.  
+
# Take out reagents from -'''20° C''' freezer and put them on ice at all times, especially DNA polymerase.  
-
# Make a master mix that contains PCR buffer, dNTP, ddH2O and DNA polymerase. Prepare enough master mix for one more sample than you need in order to compensate for pipetting errors.  Return the reagents to the -*20⁰C* freezer.  
+
# Make a master mix that contains PCR buffer, dNTP, ddH<sub>2</sub>O and DNA polymerase. Prepare enough master mix for one more sample than you need in order to compensate for pipetting errors.  Return the reagents to the -'''20° C''' freezer.  
# Transfer appropriate volume of master mix into each PCR tube.  
# Transfer appropriate volume of master mix into each PCR tube.  
# Add the correct forward and reverse primers to each sample tube.  
# Add the correct forward and reverse primers to each sample tube.  
# Add template DNA to each tube. (Negative control sample does not have template DNA)  
# Add template DNA to each tube. (Negative control sample does not have template DNA)  
# Gently vortex the samples or spin down to collect drops.
# Gently vortex the samples or spin down to collect drops.
 +
# Place the PCR tubes into the machine. Program the machine as follows:
 +
 +
<div class="aside" style="width: auto; display: inline-block; margin-top: 10px;">
 +
{|
 +
|Step
 +
|Temperature (° C)
 +
|Time (min)
 +
|Number of cycles
 +
|-
 +
|Initial Denaturation
 +
|95
 +
|1-3
 +
|1
 +
|-
 +
|Denaturation
 +
|95
 +
|0.5
 +
|20-40
 +
|-
 +
|Annealing
 +
|T<sub>m</sub> – 3
 +
|0.5
 +
|20-40
 +
|-
 +
|Extension
 +
|72
 +
|1 min/kb
 +
|20-40
 +
|-
 +
|Final Extension
 +
|72
 +
|15
 +
|1
 +
|-
 +
|Incubation
 +
|10
 +
|15
 +
|1
 +
|-
 +
|}
 +
</div>
 +
 +
<html></div></html>
 +
 +
<html><div class="section"><h2>Gel Extraction</h2></html>
 +
 +
Latest revision: '''June 3, 2010'''
 +
 +
<h3>Materials</h3>
 +
 +
''Refer to gel extraction kit''
 +
 +
<h3>Procedure</h3>
 +
 +
''Refer to gel extraction kit''
 +
 +
<html></div></html>
-
|Step|Temperature(°C)|Time(min)|Number of cycles|
+
'''[[Team:Queens-Canada/guide|Return to the Guide Hub]]'''
-
|Initial Denaturation|95|1-3|1|
+
-
|Denaturation|95|0.5|20-40|
+
-
|Annealing|T ~m~ -3|0.5|20-40|
+
-
|Extension|72|1 min/kb|20-40|
+
-
|Final Extension|72|15|1|
+
-
|Incubation|10|15|1|
+
-
====Microinjection====
+
{{:Team:Queens-Canada/foot}}

Latest revision as of 04:57, 27 October 2010

Miscellaneous Protocols

You can open and collapse protocols by clicking on their titles.

Rehydration

Latest revision: June 3, 2010

Materials

  • kit plate (from iGEM parts distribution)
  • diH2O

Procedure

  1. Using sterile technique, load 10 μL of diH2O into a p20 pipette.
  2. Pierce the foil of the correct well on the distribution plate.
  3. Firmly but carefully lower the pipette tip into the very bottom of the well.
  4. Mix the DNA with the water by slowly pumping the water in and out of the pipette tip. If done correctly, the water should turn red.
  5. Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.
  6. Write "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.
  7. Ensuring that you keep the plate level, put it into a 4° C fridge, and leave it there for one hour to allow all of the DNA to dissolve.
  8. Transfer the entire DNA solution from the well into an Eppendorf tube.

Heat Shock

Latest revision: June 3, 2010

Materials

  • 100 μL of chemical competent cells (TOP10) per transformation
  • Miniprep plasmid DNA
  • 2XTY or SOC medium
  • Antibiotic plates (according to plasmid)

Procedure

  1. Take out competent cells from -80° C and put them on ice immediately before they are needed.
  2. Add 2 μL plasmid DNA to thawed cells and mix by flicking the side of the tube. Incubate on ice for *20 minutes.
  3. Pre-warm antibiotic plates in 37° C incubator.
  4. Heat shock for 1 min 15 sec at 42° C.
  5. Place on Ice for 2 minutes.
  6. Add 500 μL 2XTY (or SOC) medium (kept at room temp.) to each tube.
  7. Shake the tubes at 37° C for 1 hour on a shaking incubator.
  8. Spread 100 μL of each transformation tube on appropriate antibiotic plates.
  9. Incubate at 37° C overnight.

Liquid Culture

Latest revision: June 3, 2010

Materials

  • Glass or plastic culture tubes
  • Growth medium containing appropriate antibiotics
  • Glass pipette tubes
  • Parafilm

Procedure

  1. Flame a glass pipette, open the bottle of medium and flame the mouth.
  2. Withdraw amount you need to fill your tubes (5 mL per tube), flame the cap and recap the bottle as quickly as possible.
  3. Remove the tube cap, flame the top of the culture tube, pipette in 5 mL, flame the top of the tube and cap it.
  4. Pick up one colony using a P20 pipette tip. Uncap the tube, flame the top, inject the tip into the tube.
  5. Incubate the tubes at 37° C overnight or until cells have reached the desired concentration. This should take between 12 and 16 hours.
  6. Seal the transformed bacteria culture plate(s) that were used with Parafilm and store in 4° C fridge.

Glycerol Stock

Latest revision: June 3, 2010

Materials

  • Overnight bacterial cell culture
  • Cryogenic screw-cap vials
  • 30% glycerol in H2O

Procedure

  1. Pipette 750 μL 30% glycerol into cryogenic vials. (Note: withdraw very slowly as glycerol is very viscous)
  2. Add 750 μL of overnight culture to each vial.
  3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
  4. Label each vial in accordance with labelling convention.
  5. Store in a freeze box in a -80° C freezer.

Miniprep

Latest revision: June 3, 2010

Materials

Refer to miniprep kit

Procedure

Refer to miniprep kit

Digestion

Latest revision: June 3, 2010

Materials

  • Digestion Buffer
  • Enzyme(s)
  • DNA
  • ddH2O

Procedure

  1. Remove the digestion buffer and enzymes from the -20° C freezer, and place on ice
  2. Add 14 µL of water to an Eppendorf tube
  3. Thaw the digestion buffer and the enzymes
  4. Add digestion buffer to tube
  5. Add DNA to tube
  6. Add enzymes to tube
  7. Incubate (time and temperature are variable and depend upon the enzymes used)

Ligation

Latest revision: June 3, 2010

Materials

Linear vector DNA 20-100 ng
Insert DNA 6:1 molar ratio of insert to vector
5X T4 ligase buffer 4 µL
T4 DNA Ligase 1 µL
Water, nuclease-free 15 µL – vector and insert vol
Total volume 20 µL

Procedure

  1. Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase
  2. Transfer 5 µL of the mastermix to each properly labeled sample tube.
  3. Add corresponding insert DNA and vector DNA into the tubes. Top the volume to 20 µl using ddH2O.
  4. Incubate one hour at 22° C (or room temperature)
  5. Heat inactivate T4 DNA ligase at 65° C for 10 min.
  6. Use up to 5 µL of the mixture for transformation of chemically competent cells.

Gel Electrophoresis

Latest revision: June 3, 2010

Materials

  • Loading dye
  • 100kb DNA ladder
  • 1% agarose gel (50 mL 1X TBE and 1 g agarose)
  • Gel box and power supply
  • Ethidium bromide stain
  • UV box/gel imager

Procedure

Making Agarose Gel

Weigh out 0.5 g of agarose and add to a 250 mL Erlenmeyer flask.

  1. Add 50 mL 1X TBE to the flask and mix by swirling.
  2. Microwave TBE agarose until the solution becomes clear and obtains a uniform consistency. (First microwave for 1 min and then for 30 sec intervals. DO NOT allow the solution to boil over in the microwave).
  3. Use glove to remove flask from the microwave. Allow flask to cool on the lab bench for 5 min (But do not wait until the gel starts to polymerize)
  4. Take out EtBr from -20° C freezer. Add 3 μL to 50 mL TBE agarose and swirl the flask to mix. Return EtBr to the freezer immediately after use. (Note: EtBr is a carcinogen and a mutagen. Always use glove and lab coat, if available, to handle things contaminated with EtBr.)
  5. Carefully pour TBE agarose into the casting tray to avoid bubbles. Make sure the tray is placed on a flat surface. Insert comb into the TAE agarose gel.
  6. Let the gel polymerize for 20 min.

Sample Preparation

  1. Add appropriate amount of loading dye such that the mixture of loading dye and sample contains 1X concentration of dye. If using 5X dye, use 4 parts sample and 1 part dye.
  2. Use appropriate volume of ladder (depends on the ladder used).

Electrophoresis

  1. Once the gel is solidified, remove the comb carefully and place the casting tray in the gel box. Make sure the wells point towards the black (negative) electrode.
  2. Fill the gel box with TBE until the entire gel is immersed in solution.
  3. Load prepared samples into the wells. Slowly pull out the pipette tip from the well before releasing the piston of the pipette. This avoids inserting bubbles into the wells, which will disturb the sample.
  4. Close the lid of the gel box. Run the gel at 100 V constant voltage for 1 hour.

Imaging

  1. Turn off the gel box power supply.
  2. Transport the gel in a plastic box to the dark room. Bring two sets of gloves if you are doing this by yourself, because you cannot touch the computer mouse with EtBr contaminated gloves. The dark room key (with attached USB key) is in the top drawer of the gel box bench.
  3. Log in to the computer.
  4. Open Genesnap. Click the big green button on the left to take a picture, and manipulate it with the sliders on the right.
  5. Print out the gel picture, label each lane, and paste it into your lab book.
  6. Save the picture (in .sgd and .jpg formats) in the QGEM folder on the USB stick.
  7. Upload the picture to BaseCamp.
  8. Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door.

PCR

Latest revision: June 3, 2010

Materials

10X PCR buffer (w/ MgCl2) 5.0 μL
25 mM dNTP mix 5.0 μL
10μM ddH2O 33.5 μL
Forward primer (10μM) 2.0 μL
Reverse primer (10μM) 2.0 μL
Template DNA 2.0 μL
DNA polymerase (eg. Taq) 0.5 μL (1.25 unit)
Total volume 50 μL

Procedure

  1. Label PCR tubes with sample names. Always include a negative control sample, which lacks template DNA.
  2. Take out reagents from -20° C freezer and put them on ice at all times, especially DNA polymerase.
  3. Make a master mix that contains PCR buffer, dNTP, ddH2O and DNA polymerase. Prepare enough master mix for one more sample than you need in order to compensate for pipetting errors. Return the reagents to the -20° C freezer.
  4. Transfer appropriate volume of master mix into each PCR tube.
  5. Add the correct forward and reverse primers to each sample tube.
  6. Add template DNA to each tube. (Negative control sample does not have template DNA)
  7. Gently vortex the samples or spin down to collect drops.
  8. Place the PCR tubes into the machine. Program the machine as follows:
Step Temperature (° C) Time (min) Number of cycles
Initial Denaturation 95 1-3 1
Denaturation 95 0.5 20-40
Annealing Tm – 3 0.5 20-40
Extension 72 1 min/kb 20-40
Final Extension 72 15 1
Incubation 10 15 1

Gel Extraction

Latest revision: June 3, 2010

Materials

Refer to gel extraction kit

Procedure

Refer to gel extraction kit

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