Difference between revisions of "Team:UMaryland/protocols"

 
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−
<div id='layer1'>
+
<div id='cover'>
−
<div id='contentbox'>
+
<div id='bar'>
 +
<p style="font-size:64px"><b>Protocols</b>
 +
</div>
 +
</div>
  
−
<!--Attention! If you are not part of the website team, you are NOT allowed to touch anything above this line without the express permission of Best Kohai.-->
 
  
−
<!--______________ADD CONTENT BELOW______________-->
+
<div id='yolo'>
 +
<div id='sidemenu'>
 +
<ul>
  
−
Protocols
+
<li><a href="#Miniprep" class="smoothScroll"><span>Miniprep</span></a></li>
−
Protocols
+
−
Miniprep:
+
−
Materials:
+
−
250 µL Buffer P1
+
−
250 µLBuffer P2
+
−
350 µL Buffer N3
+
−
750 µL Buffer PE
+
−
100 µL DDH2O
+
−
Procedure:
+
  
−
Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube)by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)
+
<li><a href="#Ligation" class="smoothScroll"><span>Ligation</span></a></li>
−
Resuspend pellet in 250 µL Buffer P1
+
−
Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear
+
−
Do not allow reaction to proceed for more than 5 mins
+
−
If using Lyse Blue, reagent, solution will turn blue
+
−
Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
+
−
If using Lyse blue, the solution will turn colorless
+
−
Centrifuge for 10 mins at 13000 rpm
+
−
Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting
+
−
Centrifuge for 60 secs and discard flow through
+
−
Was the Q1A prep column with 750 µL Buffer PE
+
−
Centrifuge for 60 secs and discard flow through
+
−
Centrifuge for 60 secs to remove residual wash buffer
+
−
Place Q1A prep column in a clean 1.5 mL microcentrifuge tube
+
−
To elute DNA, add 50 µL DDH2O to the center of Q1A prep column
+
−
Let stand for 1 min and centrifuge for 1 min
+
−
Repeat steps 12 and 13
+
  
−
Ligation:
+
<li><a href="#Transformation" class="smoothScroll"><span>Transformation</span></a></li>
−
2 µL PSBIA3 digest
+
−
2 µL upstream digest (pBAD/ sRNBC)
+
−
2 µLdownstream digest (miraculin / const_GFP)
+
−
1 µL T4 DNA ligase
+
−
2 µL T4 DNA ligase 10x rxn buffer
+
−
Let stand 10 minutes at room temperature / no heat kill
+
−
Transformation
+
−
50 µL cells
+
−
2 µL DNA
+
−
incubate on ice for 30 minutes
+
−
heats shock at 42 for 30 seconds
+
−
incubate on ice for 5 minutes
+
−
ADD 1 mL SOC media
+
−
incubate for 1 hour at 37
+
−
plate 200 µL
+
−
incubate at 37
+
−
3A assembly
+
−
5 µL Cutsmart
+
−
.5 µL BSA
+
−
.5 µL upstream
+
−
.5 µL downstream
+
−
ADD H20 to 20 µL
+
−
place in thermocycler
+
−
37 for 30 mins
+
−
80 for 20 mins
+
  
−
Gel extraction:
+
<li><a href="#3Assembly" class="smoothScroll"><span>3A Assembly</span></a></li>
−
cut out gel portions with scalpel
+
−
weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel
+
−
Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
+
−
After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
+
−
Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)
+
−
THe color of solution relates to ph indicator in
+
  
 +
<li><a href="#REDigest" class="smoothScroll"><span>RE Digest</span></a></li>
  
 +
<li><a href="#Gibsonassembly" class="smoothScroll"><span>Gibson Assembly</span></a></li>
  
−
<!--______________ADD CONTENT ABOVE______________-->
+
<li><a href="#PCR" class="smoothScroll"><span>PCR</span></a></li>
  
 +
<li><a href="#Gelextract" class="smoothScroll"><span>Gel Extract</span></a></li>
 +
<li><a href="#FPLC" class="smoothScroll"><span>FPLC</span></a></li>
 +
</ul>
 
</div>
 
</div>
 +
 +
 +
<div id='contentbox'>
 +
<a name="Miniprep">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Miniprep</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 250 µL Buffer P1</li>
 +
  <li>- 250 µLBuffer P2</li>
 +
  <li>- 350 µL Buffer N3</li>
 +
  <li>- 750 µL Buffer PE</li>
 +
  <li>- 100 µL DDH2O</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Pellet 1 mL bacterial overnight culture 5 times ( using a 1.5 mL centrifuge tube) by centrifugation at max speed (13000 rpm) for 60 secs room temperature (15 - 25)</li>
 +
  <li>- Resuspend pellet in 250 µL Buffer P1</li>
 +
  <li>- Add 250 µLBuffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes clear </li>
 +
  <li>- Do not allow reaction to proceed for more than 5 mins</li>
 +
  <li>- If using Lyse Blue, reagent, solution will turn blue</li>
 +
  <li>- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
 +
  </li>
 +
  <li>- Centrifuge for 10 mins at 13000 rpm</li>
 +
  <li>- Apply 800 µLsupernatant from step 5 to Q1A prep 2.0 spin column by pipetting</li>
 +
  <li>- Centrifuge for 60 secs and discard flow through</li>
 +
  <li>- Wash the Q1A prep column with 750 µL Buffer PE</li>
 +
  <li>- Centrifuge for 60 secs and discard flow through</li>
 +
  <li>- Centrifuge for 60 secs to remove residual wash buffer</li>
 +
  <li>- To elute DNA, add 50 µL DDH2O to the center of Q1A prep column</li>
 +
  <li>- Let stand for 1 min and centrifuge for 1 min</li>
 +
  <li>- Repeat steps 12 and 13</li>
 +
</ul>
 +
<br>
 +
<br>
 
</div>
 
</div>
  
 +
<div id='contentbox'>
 +
<a name="Ligation">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Ligation</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 2 µL PSBIA3 digest</li>
 +
  <li>- 2 µL upstream digest (pBAD/ sRNBC)</li>
 +
  <li>- 2 µLdownstream digest (miraculin / const_GFP)</li>
 +
  <li>- 1 µL T4 DNA ligase</li>
 +
  <li>- 2 µL T4 DNA ligase 10x rxn buffer</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Combine reagents in a clean PCR tube</li>
 +
  <li>- Let stand 10 mins at room temperature/ no heat kill</li>
 +
 
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
  
 +
<div id='contentbox'>
 +
<a name="Transformation">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Transformation</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 50 µL cells (DH5alpha)</li>
 +
  <li>- 2 µL DNA </li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Add DNA to cells</li>
 +
  <li>- Incubate on ice for 30 minutes</li>
 +
  <li>- Heat shock at 42°C fro 30 seconds</li>
 +
  <li>- Add 1 mL SOC media to the cells</li>
 +
  <li>- Incubate for 60 minutes at 37°C</li>
 +
  <li>- Plate 200 µL </li>
 +
  <li>- Incubate at 37°C</li>
 +
 
 +
</ul>
 +
<br>
 +
<br>
 
</div>
 
</div>
 +
<div id='contentbox'>
 +
<a name="3Assembly">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>3A Assembly</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Materials:
 +
<ul class="a">
 +
  <li>- 5 µL CutSmart</li>
 +
  <li>- 0.5 µL BSA </li>
 +
  <li>- 0.5 µL upstream digest (DH5alpha)</li>
 +
  <li>- 0.5 µL downstream digest </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 in the thermocycler</li>
 +
  <li>- 37°C for 30 minutes</li>
 +
  <li>- 80°C for 20 minutes</li> 
 +
</ul>
 +
<br>
 +
<br>
 
</div>
 
</div>
  
 +
<div id='contentbox'>
 +
<a name="REDigest">
 +
<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'>
 +
<a name="Gibsonassembly">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Gibson 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'>
 +
<a name="PCR">
 +
<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'>
 +
<a name="Gelextract">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Gel Extract</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Cut out gel portions with scalpel</li>
 +
  <li>- Weigh gel slice in colorless tube. Add 3 volumes of QG buffer to 1 volume of gel</li>
 +
  <li>- Incubate at 50 for 10 mins until gels slice dissolves. Mix by vortexing tube every 2-3 mins during incubation to help the gel dissolve
 +
</li>
 +
  <li>- After the gel dissolves completely, check color of the mixture is yellow (it may be orange or violet)
 +
</li>
 +
  <li>- Add 10 µL of 3M sodium acetate (ph 5.0) to make solution more yellow (regardless of current solution color)</li>
 +
  <li>- The color of solution relates to ph indicator in Q3</li>
 +
  <li>- Add 1 gel volume of of isopropyl to the sample and mix. This increases yield of DNA fragments between 500 bp and 4KB</li>
 +
  <li>- Place a Q1Aquick spin column in a provided 2ml collection tube</li>
 +
  <li>- Apply sample to spin column, centrifuge for 1 min to bind dna</li>
 +
  <li>- Discard flow through</li>
 +
  <li>- Wash with .75 mL buffer PE in column, centrifuge 1 minute</li>
 +
  <li>- Place column in a clean 1.5 microcentrifuge tube</li>
 +
  <li>- 40 microliters DDH20 , let stand 1 minute, centrifuge 1 min</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
<div id='contentbox'>
 +
<a name="FPLC">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Fast Protein Liquid Chromatography</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Procedure:
 +
<ul class="a">
 +
  <li>- Centrifuge cell lysate (balance with equal mass of water) for 60 minutes at 25000 rpm</li>
 +
  <li>- Wash FPLC column with equilibration column (X2) with  syringe</li>
 +
  <li>- Wash out FLPC system with equilibration buffer</li>
 +
  <li>- Wash affinity column with equilibration column with  syringe</li>
 +
  <li>- Wash affinity  column with cobalt acetate with syringe</li>
 +
  <li>- Wash off excess cobalt with syringe</li>
 +
  <li>- Connect affinity column to FPLC system</li>
 +
  <li>- Inject supernatant</li>
 +
  <li>- Wash FPLC column with equilibration column (X2) with syringe</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
</div>
 +
</div>
  
 
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Latest revision as of 02:32, 19 September 2015