Difference between revisions of "Team:NYMU-Taipei/experimentsandresults"
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<div class="protocol"> | <div class="protocol"> | ||
<div id="2t" class="trigger"onclick="detail(event)">Colony PCR (Thermo DreamTaq®)</div> | <div id="2t" class="trigger"onclick="detail(event)">Colony PCR (Thermo DreamTaq®)</div> | ||
| + | <div id="2d" class="detail"> | ||
<ol> | <ol> | ||
<li>Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified: | <li>Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified: | ||
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</li> | </li> | ||
</ol> | </ol> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="3t" class="trigger"onclick="detail(event)">Liquid culture:</div> |
| + | <div id="3d" class="detail"> | ||
<ol> | <ol> | ||
<li>Aliquot 4 mL of LB medium in a centrifugal tube for each colony.</li> | <li>Aliquot 4 mL of LB medium in a centrifugal tube for each colony.</li> | ||
<li>Use a tip and dip it in the LB culture and swirl it around.</li> | <li>Use a tip and dip it in the LB culture and swirl it around.</li> | ||
</ol> | </ol> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | < | + | <div id="4t" class="trigger"onclick="detail(event)">Draw on 2nd time plate:</div> |
| + | <div id="4d" class="detail"> | ||
<p>Take out new plates from the fridge and divide them into smaller sections. This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check.</p> | <p>Take out new plates from the fridge and divide them into smaller sections. This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check.</p> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="5t" class="trigger"onclick="detail(event)">Making Gel for gel electrophoresis</div> |
| + | <div id="5d" class="detail"> | ||
<ol> | <ol> | ||
<li>Make either 100mL or 150mL of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)</li> | <li>Make either 100mL or 150mL of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)</li> | ||
| Line 206: | Line 213: | ||
<li>Store in 4°C and away from light.</li> | <li>Store in 4°C and away from light.</li> | ||
</ol> | </ol> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="6t" class="trigger"onclick="detail(event)">Gel electrophoresis</div> |
| + | |||
</div> | </div> | ||
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<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="7t" class="trigger"onclick="detail(event)">Plasmid Extraction</div> |
| − | <div id=" | + | <div id="7d" class="detail">ballalalla</div> |
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="8t" class="trigger"onclick="detail(event)">Plasmid Extraction</div> |
| − | <div id=" | + | <div id="8d" class="detail">ballalalla</div> |
</div> | </div> | ||
<div class="protocol"> | <div class="protocol"> | ||
| − | <div id=" | + | <div id="9t" class="trigger"onclick="detail(event)">Plasmid Extraction</div> |
| − | <div id=" | + | <div id="9d" class="detail">ballalalla</div> |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 12:19, 1 October 2018
HAIR TO STAY
Protocols
E.coli Protocols
Two-day Efficient Cloning Cycle
We used an efficient two day cloning cycle split into a "Light" day and a "Heavy" day.
Light Day
The light day consists of Colony PCR and liquid culture of colonies transformed from a previous day.
-
The 3-in-1
First, count the number of colonies you want to check. Then, do the following 3 things sequentially:
- Liquid culture
- 2nd time plate
- Colony PCR
(Use the same tip to add the template to these three things)
- Make The Gel For Electrophoresis
- Run Gel Electrophoresis To Check The Colony PCR Product
Heavy Day
The heavy day consists of:
- Previously grown plasmid extraction
- Plasmid PCR
- Gel extraction
- Digestion
- Ligation
- Transformation
Colony PCR (Thermo DreamTaq®)
- Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified:
Item uL Primer(Forward and reverse) 1 dNTP (10mM) 1 10x DreamTaq buffer 5 Taq Polymerase 0.2 ddH20 42.8 Total 50 - Select a colony using a tip or toothpick.
- Dip it in a PCR tube and swirl it around.
PCR run protocol
Temperature Time 94℃ 60s 94℃ 15s 55℃ 20s 30-35 cycles 72℃ 1kb/min + 5-10s 72℃ 300s
Liquid culture:
- Aliquot 4 mL of LB medium in a centrifugal tube for each colony.
- Use a tip and dip it in the LB culture and swirl it around.
Draw on 2nd time plate:
Take out new plates from the fridge and divide them into smaller sections. This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check.
Making Gel for gel electrophoresis
- Make either 100mL or 150mL of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)
- Measure out the relevant percentage in agarose (e.g. 1g of agarose for 100mL of 1% gel).
- Fill it up with 1xTAE buffer.
- Microwave on low, constantly taking it out every so often to swirl and mix it. Keep microwaving until clear. Make sure it is completely clear
- Cool to a temperature that is still hot but still can be held in your hand. If the gel gets too cold and starts to harden, start microwaving again until clear.
- Add 5uL of Safe-seeing dye for every 100mL of gel after cooled to the correct tempearature. Mix it well by swirling
- Pour it onto the molds quickly. Put a cover on it to block out the light.
- Wait at least 15-20 min until using it.
- Store in 4°C and away from light.
Gel electrophoresis
Plasmid Extraction
ballalalla
Plasmid Extraction
ballalalla
Plasmid Extraction
ballalalla