Difference between revisions of "Team:UFMG Brazil/Protocols"

 
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<h3>Protocols</h3>
 
<h3>Protocols</h3>
<h4>Bacteria mediums</h4>
 
  
<p> <u>*LB (Luria-Bertani) liquid medium – 300 ml </u></br>
+
<h4>PCR for IFN-β amplification</h4>
3 g tryptone</br>
+
1.5 g yeast extract</br>
+
<p><u>PCR reaction</u><br/></p>
3 g NaCl</br>
+
<p class="indent">
Complete to 300 ml with distilled water</br>
+
3 μl incubation buffer<br/>
Autoclave in a 500 ml Erlenmeyer</br>
+
3 μl primer F<br/>
Prepare 2 Erlenmeyers with 300 ml of medium each.
+
3 μl primer R<br/>
</br></p>
+
1 μl INF-β plasmidial DNA (“pUCIDT gene 1-LAP-INFB”)<br/>
 +
3 μl dNTP (10x diluted)<br/>
 +
17 μl H2O<br/>
 +
0.5 μl Taq DNA Polymerase<br/></p>
 +
 +
<p><u>Thermocycler program:</u><br/></p>
 +
<img class="centered-image" src="https://static.igem.org/mediawiki/2015/f/fc/UFMG-Brazil-Protocols-Table.png"/><br/>
  
<p> <u>*LB (Luria-Bertani) liquid medium 300 ml </u></br>
+
<h4>Agarose DNA electrophoresis gel</h4>
3 g tryptone</br>
+
1.5 g yeast extract</br>
+
<p><u>Gel materials 30 mL gel 1.2%</u><br/></p>
3 g NaCl</br>
+
4.5 g Agar (weigh directly into the Erlenmeyer)</br>
+
<ul class="disc"><li>0.36 g agarose</li>
Complete to 300 ml with distilled water</br>
+
<li>30 mL TAE Buffer (tris-acetate-EDTA)</li>
Autoclave in a 500 ml Erlenmeyer</br>
+
<li>1.5 µL Sybr Safe</li></ul><br/>
</br>
+
<u>*SOC medium – 100 ml</u>
+
<p><u>TAE Buffer materials – 1L</u><br/></p>
2g tryptone</br>
+
0.5 g yeast extract</br>
+
<ul class="disc"><li>242 g Tris Base</li>
200 µl NaCl 5 M</br>
+
<li>57.1 mL Cold Acetic Acid</li>
1 ml MgCl2 1 M</br>
+
<li>100 mL 0.5 M EDTA</li>
250 µl KCl 1 M</br>
+
<li>1 L distilled water</li></ul><br/>
10 ml MgSO4 1 M</br>
+
Complete to 100 ml with distilled water</br>
+
<p><u>Sample materials - 12 µL</u><br/></p>
Autoclave and store in bottle with lid</br>
+
 +
<ul class="disc"><li>1 kb DNA ladder</li>
 +
<li>10 µL DNA samples:</li>
 +
                <li>8 µL nuclease-free water</li>
 +
                <li>2 µL Miniprep DNA</li>
 +
<li>2 µL loading buffer</li></ul><br/>
 +
 +
<p><u>Methodology</u><br/></p>
 +
<p>Prepare the TAE buffer joining the materials in a volumetric flask, beaker or container at the stated order.<br/>
 +
 +
Add the agarose and TAE in Erlenmeyer. Mix and heat it carefully in the microwave until homogeneous and without crystals.<br/>
 +
 +
Wait the glass cooling until the temperature reaches ~60° C. Add Syber Safe and pour the solution in the appropriate gel-forming container. Insert the comb to form the wells and wait for gel solidification.<br/>
 +
 +
Remove the comb, place the gel on the running cube, and immerse gel in TAE.<br/>
 +
 +
In a microtube or paraplast tape, mix the loading buffer with the DNA and carefully apply it to the gel. Apply the 1 kb DNA Ladder to gel as well.<br/>
 +
 +
Plug the electrodes and run gel for ~40 min.<br/></p><br/>
  
<li>Preparing NaCl 5 M
+
<h4>Double digestion</h4>
<ul class="circle">
+
<li>NaCl MW: 58.44 g/mol</li>
+
<p><u>Double digestion reaction:</u><br/></p>
<li>58.44 g ---- 1 mol ---- 1000 ml</li>
+
          <p class="indent">4.8 μl Nuclease-free water<br/>
        x ------------------------- 50 ml
+
          12 μl pLP-neo miniprep DNA<br/>
        x = 2.92 g</br></li></ul>
+
          0.2 μl BSA<br/>
 +
          2 μl Multicore 10x buffer<br/>
 +
          0.5 μl XbaI enzyme<br/>
 +
          0.5 μl BamHI enzyme<br/></p>
 +
<p>Final volume: 20 μl<br/>
 +
Quick spin<br/>
 +
Incubation at 37º C for 3 h<br/>
 +
Inactivation at 56º C for 30 min<br/></p><br/>
  
<ul class="disc">
+
<h4>Ligation</h4>
 +
<p>We used the IFN-β DNA amplified by PCR for ligation with pGEM-T Easy vector System I (Promega).<br/></p>
 +
 +
<p><u>Reaction - total 10 μl</u><br/></p>
 +
          <p class="indent">5 μl 2x Rapid Ligation Buffer, T4 DNA Ligase<br/>
 +
          1 μl pGEM-T Easy vector (50 ng)<br/>
 +
          3 μl IFN-β PCR product from tube 1<br/>
 +
          1 μl T4 DNA Ligase (3 Weiss units/μl)<br/></p>
 +
 +
<p>Incubation for 1 h at room temperature. After, the ligation was incubated at 4° C, overnight.<br/></p><br/>
  
<li>2.92 g -------- 1 mol</li>
+
<h4>Bacteria mediums</h4>
        y --------------- 5 mol
+
<p><u>LB (Luria-Bertani) liquid medium – 300 ml</u><br/></p>
        y = 14.6 g</ul></br>
+
<ul class="disc"><li>3 g tryptone</li>
 +
<li>1.5 g yeast extract</li>
 +
<li>3 g NaCl</li></ul>
 +
<p>Complete to 300 ml with distilled water<br/>
 +
Autoclave in a 500 ml erlenmeyer<br/>
 +
Prepare 2 erlenmeyers with 300 ml of medium each<br/></p>
 +
 +
<p><u>LB (Luria-Bertani) Agar medium – 300 ml</u><br/></p>
 +
<ul class="disc"><li>3 g tryptone</li>
 +
<li>1.5 g yeast extract</li>
 +
<li>3 g NaCl</li>
 +
<li>4.5 g Agar (weigh directly into the erlenmeyer)</li></ul>
 +
<p>Complete to 300 ml with distilled water<br/>
 +
Autoclave in a 500 ml erlenmeyer<br/></p>
 +
 +
<p><u>SOC medium – 100 ml</u><br/></p>
 +
<ul class="disc"><li>2 g tryptone</li>
 +
<li>0.5 g yeast extract</li>
 +
<li>200 µl NaCl 5 M</li>
 +
<li>1 ml MgCl2 1 M</li>
 +
<li>250 µl KCl 1 M</li>
 +
<li>10 ml MgSO4 1 M</li></ul>
 +
<p>Complete to 100 ml with distilled water<br/>
 +
Autoclave and store in bottle with lid<br/></p>
  
Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water.</br></br>
+
<p><u>Preparing NaCl 5 M</u><br/></p>
 +
<p class="formula">NaCl MW: 58.44 g/mol<br/>
 +
58.44 g ---- 1 mol ---- 1000 ml<br/>
 +
          x ------------------------- 50 ml<br/>
 +
          x = 2.92 g<br/>
 +
2.92 g -------- 1 mol<br/>
 +
          y --------------- 5 mol<br/>
 +
          y = 14.6 g<br/>
 +
Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water<br/></p>
 +
     
 +
<p><u>Preparing MgCl2 1 M</u><br/></p>
 +
<p class="formula">MgCl2 MW: 95.211 g/mol<br/>
 +
95.21 g ---- 1 mol ---- 1000 ml<br/>
 +
          x ------------------------- 50 ml<br/>
 +
          x = 4.7 g<br/>
 +
Put 4.7 g MgCl2 into a 50 ml falcon tube and complete to 50 ml with distilled water<br/></p>
 +
       
 +
<p><u>Preparing KCl 1 M</u><br/></p>
 +
<p class="formula">KCl MW: 74.5513 g/mol<br/>
 +
74.55 g ---- 1 mol ---- 1000 ml<br/>
 +
          x ------------------------- 50 ml<br/>
 +
          x = 3.72 g<br/>
 +
Put 3.72 g KCl into a 50 ml falcon tube and complete to 50 ml with distilled water<br/></p>
  
<li>Preparing MgCl2 1 M
 
<ul class="circle">
 
<li>MgCl2 MW: 95.211 g/mol</li>
 
<li>95.21 g ---- 1 mol ---- 1000 ml</li>
 
        x ------------------------- 50 ml
 
        x = 4.7 g
 
<br>Put 4.7 g MgCl2 into a 50 ml falcon tube and complete to 50 ml with distilled water.</ul></br>
 
  
<li>Preparing KCl 1 M
+
<h4>TOP10 <i>E.coli</i> bacteria chemo competent transformation</h4>
<ul class="circle">
+
<p>2 μl of pUCIDT gene 1-LAP-INFβ plasmid in 100 μl of TOP10 bacteria<br/>
<li>KCl MW: 74.5513 g/mol</li>
+
30 min ice incubation<br/>
<li>74.55 g ---- 1 mol ---- 1000 ml</li>
+
90 s incubation in 42º C water bath;<br/>
        x ------------------------- 50 ml
+
2 min ice incubation;<br/>
        x = 3.72 g
+
Carefully add 0.9 ml of SOC medium;<br/>
</br>Put 3.72 g KCl into a 50 ml falcon tube and complete to 50 ml with distilled water.
+
1 h incubation in 37° C and 120 rpm of rotation;<br/>
</ul>
+
2 bacterial plating of 100 μl of transformation (37° C, 16 h)<br/>
</br>
+
Each plate was made with 30 ml of LB agar and 30 μl of ampicillin 100 mg/ml<br/></p>
              <h4>Bacteria planting</h4>
+
<p>Materials used</p></br>
+
  
<ul class="disc">
+
 
  <li>Erlenmeyer flask and sterilized becker</li>
+
<h4>Bacterial plating</h4>
  <li>LB Agar</li>
+
<p><u>Materials used</u><br/></p>
  <li>Sterile petri dishes</li>
+
<ul class="disc"><li>Erlenmeyer flask and sterilized becker</li>
 +
<li>LB Agar</li>
 +
<li>Sterile petri dishes</li>
 
<li>Antibiotic</li>
 
<li>Antibiotic</li>
 
<li>Bunsen burner</li>
 
<li>Bunsen burner</li>
<li>70% Alcohol</li>
+
<li>70% Ethanol</li>
 
<li>Platinum strap</li>
 
<li>Platinum strap</li>
<li>Clones</li>
+
<li>Clones</li></ul><br/>
</ul> </br>
+
<h4>Methods</h4>
+
<p><u>Methods</u><br/></p>
<p>Heat LB Agar in the microwave. Let it cool near the flame.</p>
+
<p>Heat LB Agar in the microwave. Let it cool near the flame.<br/>
<p>Pour between 15mL and 30mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix.</p>
+
Pour between 15 mL and 30 mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix.<br/>
<p>Pour the medium in the Petri dish and let it solidify near the flame.</p></br>
+
Pour the medium in the Petri dish and let it solidify near the flame.<br/>
<p>Pipet the desired volume of clone on the plate. Spread the bacterial suspension throughout the petri dish homogeneously.</p>
+
<p>Cover the plate and incubate it at 37 °C for 12-16.</p>
+
Pipet the desired volume of clone on the plate. Spread the bacterial suspension throughout the Petri dish homogeneously.<br/>
<p>Standard antibiotics concentration used:</p></br>
+
Cover the plate and incubate it at 37° C for 12-16 h<br/></p>
 +
 +
<p>Standard antibiotics concentration used:<br/></p>
 +
<ul class="disc"><li>Ampicillin: 10 µl/ml</li>
 +
<li>Chloramphenicol: 1.2 µL/​​mL</li></ul><br/>
  
<ul class="disc">
 
  <li>Ampicillin - 10 µl / ml</li>
 
  <li>Chloramphenicol -1.2 µL / ​​mL </li>
 
</ul>
 
<h4>Double digestion</h4>
 
  
<p>Double Digestion reaction:</br></p>
+
<h4>Pre-inoculum and inoculum</h4>
 
+
<p><u>Materials used</u><br/></p>
<table align="center" style="width:50%">
+
<ul class="disc"><li>Sterilized tips</li>
  <tr>
+
<li>Pipettes</li>
    <td>Nuclease-free water    </td>
+
<li>Antibiotic</li>
    <td>4,8ul</td>
+
<li>Petri dishes containing isolated bacterial colonies</li>
    </tr>
+
<li>Sterilized test-tubes</li>
  <tr>
+
<li>LB Liquid medium</li>
    <td>pLP-neo miniprep DNA  </td>
+
<li>Bunsen burner</li></ul><br/>
    <td>12ul</td>
+
    </tr>
+
<p><u>Methodology</u><br/></p>
  <tr>
+
<ul class="disc"><li>Add 4 ml of LB liquid medium to each test-tube and the correspondent antibiotic ratio</li>
    <td>BSA</td>
+
<li>With toothpick or sterilized tip, remove an isolated colony</li>
    <td>0,2ul</td>
+
<li>Place the toothpick/tip inside the test-tube, without touching it sides</li>
    </tr>
+
<li>Leave the tubes overnight at 37° C for 16 h in the shaker</li>
<tr>
+
<li>After incubation, remove the tubes from the shaker. Medium must be turbid, indicating bacterial growth</li>
    <td>Multicore 10x buffer</td>
+
<li>Pour the contents of the test-tubes on a larger recipient and complete volume to 20 ml with liquid LB medium</li>
    <td>2ul</td>
+
<li>Repeat the incubation as above and remove the inserted plasmid through miniprep or maxiprep</li></ul><br/>
    </tr>
+
<tr>
+
    <td>XbaI enzyme</td>
+
    <td>0,5ul</td>
+
    </tr>
+
<tr>
+
    <td>bamHI enzyme</td>
+
    <td>0,5ul</td>
+
    </tr>
+
</table>
+
 
+
<p>Final volume: 20ul.</p>
+
 
+
<p>Quick spin</p>
+
<p>Incubation at 37ºC for 3 hours.</p>
+
<p>Inactivation at 56ºC for 30 minutes.</p>
+
 
+
            <h4>Agarose DNA electrophoresis gel</h4>
+
<p><b>Gel materials – 30mL gel 1.2%</b></p>
+
 
+
<ul class="disc">
+
  <li>0.36g agarose</li>
+
  <li>30mL TAE Buffer (tris-acetate-EDTA)</li>
+
  <li>1.5µL Sybr Safe</li>
+
</ul>  </br>
+
 
+
<p><b>TAE Buffer materials – 1L</b></p>
+
<ul class="disc">
+
  <li>242g Tris Base</li>
+
  <li>57.1 mL Cold Acetic Acid</li>
+
  <li>100mL 0.5M EDTA</li>
+
<li>1L distilled water</li></br>
+
</ul>
+
  
<p><b>Sample materials - 12µL</b></p>
+
<ul class="disc">
+
<h4>Glycerol Clone Storage</h4>
  <li>1kb DNA ladder</li>
+
<p><u>Materials used</u><br/></p>
  <li>10µL DNA samples:</li>
+
<ul class="disc"><li>50% Glycerol</li>
<ul class="circle"> <li>8µL nuclease-free water</li></ul>
+
<li>Sterilized microtubes</li>
<ul class="circle"> <li>2µL Miniprep DNA</li></ul>
+
<li>Sterilized tips</li>
  <li>2µL loading buffer</li>
+
<li>Pipettes</li>
</ul>
+
<li>Bacteria in liquid medium containing DNA clone</li></ul><br/>
 +
 +
<p><u>Methods</u><br/></p>
 +
<p>Homogenize bacteria medium by inversion.<br/>
 +
Near the flame, transfer 170 µL of bacterial culture to a sterile microtube.<br/>
 +
Add 30 µl of 50% glycerol and homogenize.<br/>
 +
Place the glycerol clones in a -20° C freezer for ~20 h and then transfer them to a - 80° C freezer.<br/></p><br/>
  
 
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{{UFMG_Brazil/contentbottom}}
 
{{UFMG_Brazil/contentbottom}}

Latest revision as of 03:42, 19 September 2015




Project

Overview

Problem and
Solution

Chassis

Devices and
kill switch

Lab Work

Safety

Notebook

Protocols

Results

Modeling

Practices

Overview

Integrated Human
Practices

Public
Engagement

Synenergene

Overview

Application
Scenarios

Techno-moral

Team

Our Team

Attributions

Collaborations

Sponsors




Protocols

PCR for IFN-β amplification

PCR reaction

3 μl incubation buffer
3 μl primer F
3 μl primer R
1 μl INF-β plasmidial DNA (“pUCIDT gene 1-LAP-INFB”)
3 μl dNTP (10x diluted)
17 μl H2O
0.5 μl Taq DNA Polymerase

Thermocycler program:


Agarose DNA electrophoresis gel

Gel materials – 30 mL gel 1.2%

  • 0.36 g agarose
  • 30 mL TAE Buffer (tris-acetate-EDTA)
  • 1.5 µL Sybr Safe

TAE Buffer materials – 1L

  • 242 g Tris Base
  • 57.1 mL Cold Acetic Acid
  • 100 mL 0.5 M EDTA
  • 1 L distilled water

Sample materials - 12 µL

  • 1 kb DNA ladder
  • 10 µL DNA samples:
  • 8 µL nuclease-free water
  • 2 µL Miniprep DNA
  • 2 µL loading buffer

Methodology

Prepare the TAE buffer joining the materials in a volumetric flask, beaker or container at the stated order.
Add the agarose and TAE in Erlenmeyer. Mix and heat it carefully in the microwave until homogeneous and without crystals.
Wait the glass cooling until the temperature reaches ~60° C. Add Syber Safe and pour the solution in the appropriate gel-forming container. Insert the comb to form the wells and wait for gel solidification.
Remove the comb, place the gel on the running cube, and immerse gel in TAE.
In a microtube or paraplast tape, mix the loading buffer with the DNA and carefully apply it to the gel. Apply the 1 kb DNA Ladder to gel as well.
Plug the electrodes and run gel for ~40 min.


Double digestion

Double digestion reaction:

4.8 μl Nuclease-free water
12 μl pLP-neo miniprep DNA
0.2 μl BSA
2 μl Multicore 10x buffer
0.5 μl XbaI enzyme
0.5 μl BamHI enzyme

Final volume: 20 μl
Quick spin
Incubation at 37º C for 3 h
Inactivation at 56º C for 30 min


Ligation

We used the IFN-β DNA amplified by PCR for ligation with pGEM-T Easy vector System I (Promega).

Reaction - total 10 μl

5 μl 2x Rapid Ligation Buffer, T4 DNA Ligase
1 μl pGEM-T Easy vector (50 ng)
3 μl IFN-β PCR product from tube 1
1 μl T4 DNA Ligase (3 Weiss units/μl)

Incubation for 1 h at room temperature. After, the ligation was incubated at 4° C, overnight.


Bacteria mediums

LB (Luria-Bertani) liquid medium – 300 ml

  • 3 g tryptone
  • 1.5 g yeast extract
  • 3 g NaCl

Complete to 300 ml with distilled water
Autoclave in a 500 ml erlenmeyer
Prepare 2 erlenmeyers with 300 ml of medium each

LB (Luria-Bertani) Agar medium – 300 ml

  • 3 g tryptone
  • 1.5 g yeast extract
  • 3 g NaCl
  • 4.5 g Agar (weigh directly into the erlenmeyer)

Complete to 300 ml with distilled water
Autoclave in a 500 ml erlenmeyer

SOC medium – 100 ml

  • 2 g tryptone
  • 0.5 g yeast extract
  • 200 µl NaCl 5 M
  • 1 ml MgCl2 1 M
  • 250 µl KCl 1 M
  • 10 ml MgSO4 1 M

Complete to 100 ml with distilled water
Autoclave and store in bottle with lid

Preparing NaCl 5 M

NaCl MW: 58.44 g/mol
58.44 g ---- 1 mol ---- 1000 ml
x ------------------------- 50 ml
x = 2.92 g
2.92 g -------- 1 mol
y --------------- 5 mol
y = 14.6 g
Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water

Preparing MgCl2 1 M

MgCl2 MW: 95.211 g/mol
95.21 g ---- 1 mol ---- 1000 ml
x ------------------------- 50 ml
x = 4.7 g
Put 4.7 g MgCl2 into a 50 ml falcon tube and complete to 50 ml with distilled water

Preparing KCl 1 M

KCl MW: 74.5513 g/mol
74.55 g ---- 1 mol ---- 1000 ml
x ------------------------- 50 ml
x = 3.72 g
Put 3.72 g KCl into a 50 ml falcon tube and complete to 50 ml with distilled water

TOP10 E.coli bacteria chemo competent transformation

2 μl of pUCIDT gene 1-LAP-INFβ plasmid in 100 μl of TOP10 bacteria
30 min ice incubation
90 s incubation in 42º C water bath;
2 min ice incubation;
Carefully add 0.9 ml of SOC medium;
1 h incubation in 37° C and 120 rpm of rotation;
2 bacterial plating of 100 μl of transformation (37° C, 16 h)
Each plate was made with 30 ml of LB agar and 30 μl of ampicillin 100 mg/ml

Bacterial plating

Materials used

  • Erlenmeyer flask and sterilized becker
  • LB Agar
  • Sterile petri dishes
  • Antibiotic
  • Bunsen burner
  • 70% Ethanol
  • Platinum strap
  • Clones

Methods

Heat LB Agar in the microwave. Let it cool near the flame.
Pour between 15 mL and 30 mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix.
Pour the medium in the Petri dish and let it solidify near the flame.
Pipet the desired volume of clone on the plate. Spread the bacterial suspension throughout the Petri dish homogeneously.
Cover the plate and incubate it at 37° C for 12-16 h

Standard antibiotics concentration used:

  • Ampicillin: 10 µl/ml
  • Chloramphenicol: 1.2 µL/​​mL

Pre-inoculum and inoculum

Materials used

  • Sterilized tips
  • Pipettes
  • Antibiotic
  • Petri dishes containing isolated bacterial colonies
  • Sterilized test-tubes
  • LB Liquid medium
  • Bunsen burner

Methodology

  • Add 4 ml of LB liquid medium to each test-tube and the correspondent antibiotic ratio
  • With toothpick or sterilized tip, remove an isolated colony
  • Place the toothpick/tip inside the test-tube, without touching it sides
  • Leave the tubes overnight at 37° C for 16 h in the shaker
  • After incubation, remove the tubes from the shaker. Medium must be turbid, indicating bacterial growth
  • Pour the contents of the test-tubes on a larger recipient and complete volume to 20 ml with liquid LB medium
  • Repeat the incubation as above and remove the inserted plasmid through miniprep or maxiprep

Glycerol Clone Storage

Materials used

  • 50% Glycerol
  • Sterilized microtubes
  • Sterilized tips
  • Pipettes
  • Bacteria in liquid medium containing DNA clone

Methods

Homogenize bacteria medium by inversion.
Near the flame, transfer 170 µL of bacterial culture to a sterile microtube.
Add 30 µl of 50% glycerol and homogenize.
Place the glycerol clones in a -20° C freezer for ~20 h and then transfer them to a - 80° C freezer.