Difference between revisions of "Team:UMaryland/protocols"

Line 204: Line 204:
 
   <li>- Add DNA to cells</li>
 
   <li>- Add DNA to cells</li>
 
   <li>- Incubate on ice for 30 minutes</li>
 
   <li>- Incubate on ice for 30 minutes</li>
−
   <li>- Heat shock at 42° fro 30 seconds</li>
+
   <li>- Heat shock at 42°C fro 30 seconds</li>
 
   <li>- Add 1 mL SOC media to the cells</li>
 
   <li>- Add 1 mL SOC media to the cells</li>
 
   <li>- Incubate for 60 minutes at 37°</li>
 
   <li>- Incubate for 60 minutes at 37°</li>
 
   <li>- Plate 200 µL </li>
 
   <li>- Plate 200 µL </li>
−
   <li>- Incubate at 37°</li>
+
   <li>- Incubate at 37°C</li>
 
    
 
    
 
</ul>
 
</ul>
Line 224: Line 224:
 
   <li>- 0.5 µL upstream digest (DH5alpha)</li>
 
   <li>- 0.5 µL upstream digest (DH5alpha)</li>
 
   <li>- 0.5 µL downstream digest </li>
 
   <li>- 0.5 µL downstream digest </li>
−
   <li>- Add DDH2O to 20 µL (DH5alpha)</li>
+
   <li>- Add DDH2O to 20 µL</li>
 
</ul>
 
</ul>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
Line 231: Line 231:
 
   <li>- Combine reagents in a PCR tube</li>
 
   <li>- Combine reagents in a PCR tube</li>
 
   <li>- Place in the thermocycler</li>
 
   <li>- Place in the thermocycler</li>
−
   <li>- 37° for 30 minutes</li>
+
   <li>- 37°C for 30 minutes</li>
−
   <li>- 80° for 20 minutes</li>   
+
   <li>- 80°C for 20 minutes</li>   
 
</ul>
 
</ul>
 
<br>
 
<br>
Line 238: Line 238:
 
</div>
 
</div>
  
 +
<div id='contentbox'>
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>RE Digest</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 1 µg DNA</li>
 +
  <li>- 5 µL 10X NEBuffer </li>
 +
  <li>- Add DDH2O to 20 µL</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Incubate for 1 hour</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Giscon Assembly</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 0.02-0.5 pmols of DNA</li>
 +
  <li>- 10 µL Gibson Assembly MasterMix (2X) </li>
 +
  <li>- Add DDH2O to 20 µL</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Place tube in thermocycler for 15 minutes at 50°C</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>PCR</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 4 µL Phusion HF/GC Buffer</li>
 +
  <li>- 0.4 µL 10 mM dNTPs</li>
 +
  <li>- 1 µL 10 µM forward primer</li>
 +
  <li>- 1 µL 10 µM forward primer</li>
 +
  <li>- 0.2 µL Phusion DNA polymerase</li>
 +
  <li>- (OPTIONAL) 0.6 µL DMSO</li>
 +
  <li>- <250 ng Template DNA </li>
 +
  <li>- Add DDH2O to 20 µL </li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a PCR tube</li>
 +
  <li>- Place tube in thermocycler for:</li> 
 +
  <li>- Initial Denaturation: 98° C for 30 seconds</li> 
 +
  <li>- 25-35 cycles: 98°C for 5-10 seconds, 45-72°C for 10-30 seconds and 72°C for 15-30 seconds per kb</li> 
 +
  <li>- Final Extension: 72°C</li> 
 +
  <li>- Hold 4-10°C</li> 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 
<div id='contentbox'>
 
<div id='contentbox'>
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">

Revision as of 13:42, 18 September 2015