Difference between revisions of "Team:UFMG Brazil/Protocols"
| Line 10: | Line 10: | ||
3 g NaCl</br> | 3 g NaCl</br> | ||
Complete to 300 ml with distilled water</br> | Complete to 300 ml with distilled water</br> | ||
| − | Autoclave in a 500 ml | + | Autoclave in a 500 ml Erlenmeyer</br> |
| − | Prepare 2 | + | Prepare 2 Erlenmeyers with 300 ml of medium each. |
</br></p> | </br></p> | ||
| Line 18: | Line 18: | ||
1.5 g yeast extract</br> | 1.5 g yeast extract</br> | ||
3 g NaCl</br> | 3 g NaCl</br> | ||
| − | 4.5 g Agar (weigh directly into the | + | 4.5 g Agar (weigh directly into the Erlenmeyer)</br> |
Complete to 300 ml with distilled water</br> | Complete to 300 ml with distilled water</br> | ||
| − | Autoclave in a 500 ml | + | Autoclave in a 500 ml Erlenmeyer</br> |
</br> | </br> | ||
<u>*SOC medium – 100 ml</u> | <u>*SOC medium – 100 ml</u> | ||
| Line 45: | Line 45: | ||
y = 14.6 g</ul></br> | y = 14.6 g</ul></br> | ||
| − | Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water</br></br> | + | Put 14.6 g NaCl into a 50 ml falcon tube and complete to 50 ml with distilled water.</br></br> |
<li>Preparing MgCl2 1 M | <li>Preparing MgCl2 1 M | ||
| Line 61: | Line 61: | ||
x ------------------------- 50 ml | x ------------------------- 50 ml | ||
x = 3.72 g | 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>Put 3.72 g KCl into a 50 ml falcon tube and complete to 50 ml with distilled water. |
</ul> | </ul> | ||
</br> | </br> | ||
| Line 79: | Line 79: | ||
<h4>Methods</h4> | <h4>Methods</h4> | ||
<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.</p> | ||
| − | <p>Pour between 15mL and 30mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix</p> | + | <p>Pour between 15mL and 30mL of LB Agar in an Erlenmeyer. Add the antibiotic in the right proportion and mix.</p> |
<p>Pour the medium in the Petri dish and let it solidify near the flame.</p></br> | <p>Pour the medium in the Petri dish and let it solidify near the flame.</p></br> | ||
<p>Pipet the desired volume of clone on the plate. Spread the bacterial suspension throughout the petri dish homogeneously.</p> | <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> | + | <p>Cover the plate and incubate it at 37 °C for 12-16.</p> |
<p>Standard antibiotics concentration used:</p></br> | <p>Standard antibiotics concentration used:</p></br> | ||
| Line 123: | Line 123: | ||
<p>Quick spin</p> | <p>Quick spin</p> | ||
| − | <p>Incubation at 37ºC for 3 hours.</p | + | <p>Incubation at 37ºC for 3 hours.</p> |
| − | <p>Inactivation at 56ºC for 30 minutes</p | + | <p>Inactivation at 56ºC for 30 minutes.</p> |
<h4>Agarose DNA electrophoresis gel</h4> | <h4>Agarose DNA electrophoresis gel</h4> | ||
| − | <p><b>Gel materials – 30mL gel 1.2%</b></p | + | <p><b>Gel materials – 30mL gel 1.2%</b></p> |
<ul class="disc"> | <ul class="disc"> | ||
| Line 133: | Line 133: | ||
<li>30mL TAE Buffer (tris-acetate-EDTA)</li> | <li>30mL TAE Buffer (tris-acetate-EDTA)</li> | ||
<li>1.5µL Sybr Safe</li> | <li>1.5µL Sybr Safe</li> | ||
| − | </ul> | + | </ul> </br> |
| − | <p><b>TAE Buffer materials – 1L</b></p | + | |
| + | <p><b>TAE Buffer materials – 1L</b></p> | ||
<ul class="disc"> | <ul class="disc"> | ||
<li>242g Tris Base</li> | <li>242g Tris Base</li> | ||
<li>57.1 mL Cold Acetic Acid</li> | <li>57.1 mL Cold Acetic Acid</li> | ||
<li>100mL 0.5M EDTA</li> | <li>100mL 0.5M EDTA</li> | ||
| − | <li>1L distilled water</li> | + | <li>1L distilled water</li></br> |
</ul> | </ul> | ||
| − | <p><b>Sample materials - 12µL</b></p | + | <p><b>Sample materials - 12µL</b></p> |
<ul class="disc"> | <ul class="disc"> | ||
<li>1kb DNA ladder</li> | <li>1kb DNA ladder</li> | ||
<li>10µL DNA samples:</li> | <li>10µL DNA samples:</li> | ||
| − | <ul class="circle"> 8µL nuclease-free water</ul> | + | <ul class="circle"> <li>8µL nuclease-free water</li></ul> |
| − | <ul> 2µL Miniprep DNA</ul> | + | <ul class="circle"> <li>2µL Miniprep DNA</li></ul> |
<li>2µL loading buffer</li> | <li>2µL loading buffer</li> | ||
</ul> | </ul> | ||
Revision as of 17:56, 17 September 2015
Project
Lab Work
Modeling
Practices
Synenergene
Team
Protocols
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) liquid 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 2g 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
- 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
- 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.
- 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.
Bacteria planting
Materials used
- Erlenmeyer flask and sterilized becker
- LB Agar
- Sterile petri dishes
- Antibiotic
- Bunsen burner
- 70% Alcohol
- Platinum strap
- Clones
Methods
Heat LB Agar in the microwave. Let it cool near the flame.
Pour between 15mL and 30mL 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.
Standard antibiotics concentration used:
- Ampicillin - 10 µl / ml
- Chloramphenicol -1.2 µL / mL
Double digestion
Double Digestion reaction:
| Nuclease-free water | 4,8ul |
| pLP-neo miniprep DNA | 12ul |
| BSA | 0,2ul |
| Multicore 10x buffer | 2ul |
| XbaI enzyme | 0,5ul |
| bamHI enzyme | 0,5ul |
Final volume: 20ul.
Quick spin
Incubation at 37ºC for 3 hours.
Inactivation at 56ºC for 30 minutes.
Agarose DNA electrophoresis gel
Gel materials – 30mL gel 1.2%
- 0.36g agarose
- 30mL TAE Buffer (tris-acetate-EDTA)
- 1.5µL Sybr Safe
TAE Buffer materials – 1L
- 242g Tris Base
- 57.1 mL Cold Acetic Acid
- 100mL 0.5M EDTA
- 1L distilled water
Sample materials - 12µL
- 1kb DNA ladder
- 10µL DNA samples:
- 8µL nuclease-free water
- 2µL Miniprep DNA
- 2µL loading buffer
