Difference between revisions of "Team:NEU China B/protocols"

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{{NEU_China_B}}
 
{{NEU_China_B}}
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<div class="text-center">
 
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<hr>
 
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<h1>NEU-CHINA-B iGEM2018</h1>
 
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<hr>
 
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<h3>Protocols</h3>
 
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<hr>
 
−
<h4>Contacts</h4>
 
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</div>
 
  
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<div class="panel-group content-group-lg" id="accordion1">
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<img class="center-block" src='data:img/jpg;base64,R0lGODlhugBiAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAAAAAC6
−
<div class="panel panel-white">
+
AGIAhwAAAAAAAAAAMwAAZgAAmQAAzAAA/wAzAAAzMwAzZgAzmQAzzAAz/wBmAABmMwBmZgBmmQBm
−
<div class="panel-heading">
+
zABm/wCZAACZMwCZZgCZmQCZzACZ/wDMAADMMwDMZgDMmQDMzADM/wD/AAD/MwD/ZgD/mQD/zAD/
−
<h6 class="panel-title">
+
/zMAADMAMzMAZjMAmTMAzDMA/zMzADMzMzMzZjMzmTMzzDMz/zNmADNmMzNmZjNmmTNmzDNm/zOZ
−
<a data-toggle="collapse" data-parent="#accordion1" href="#accordion-group1">Western blot</a>
+
ADOZMzOZZjOZmTOZzDOZ/zPMADPMMzPMZjPMmTPMzDPM/zP/ADP/MzP/ZjP/mTP/zDP//2YAAGYA
−
</h6>
+
M2YAZmYAmWYAzGYA/2YzAGYzM2YzZmYzmWYzzGYz/2ZmAGZmM2ZmZmZmmWZmzGZm/2aZAGaZM2aZ
−
</div>
+
ZmaZmWaZzGaZ/2bMAGbMM2bMZmbMmWbMzGbM/2b/AGb/M2b/Zmb/mWb/zGb//5kAAJkAM5kAZpkA
−
<div id="accordion-group1" class="panel-collapse collapse in">
+
mZkAzJkA/5kzAJkzM5kzZpkzmZkzzJkz/5lmAJlmM5lmZplmmZlmzJlm/5mZAJmZM5mZZpmZmZmZ
−
<div class="panel-body">
+
zJmZ/5nMAJnMM5nMZpnMmZnMzJnM/5n/AJn/M5n/Zpn/mZn/zJn//8wAAMwAM8wAZswAmcwAzMwA
−
<div class="text-center">
+
/8wzAMwzM8wzZswzmcwzzMwz/8xmAMxmM8xmZsxmmcxmzMxm/8yZAMyZM8yZZsyZmcyZzMyZ/8zM
−
<h1>
+
AMzMM8zMZszMmczMzMzM/8z/AMz/M8z/Zsz/mcz/zMz///8AAP8AM/8AZv8Amf8AzP8A//8zAP8z
−
Western blot
+
M/8zZv8zmf8zzP8z//9mAP9mM/9mZv9mmf9mzP9m//+ZAP+ZM/+ZZv+Zmf+ZzP+Z///MAP/MM//M
−
</h1>
+
Zv/Mmf/MzP/M////AP//M///Zv//mf//zP///wECAwECAwECAwECAwECAwECAwECAwECAwECAwEC
−
<h2>
+
AwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwEC
−
Put blot into antibody:
+
AwECAwECAwECAwECAwECAwECAwECAwECAwECAwECAwj/AAEIHEiwoMGDCBMqXFjQ1aBED1u1YkhR
−
</h2>
+
IDaBrlo93Fixo8ePIEOKHCkwkUOHEkkebHVy0EkA2CaqnEmzpk2KrRJB3MmyZiKNOwdpvEm0qFGa
−
</div>
+
LRUJTVQTpcunR6NKnarwZyKlOnPOdPXzoclBVMOKlZqzZUuSZp+6Gsu2LU2JG3cyJcnSq1y3ePN2
−
<ol>
+
rOv07EiNDpU6vai3MF6hA2kBcKhTriKIJAU1thtx8UDEhjOLHIpQ7VLAQj07pBs67Vm1AmVadilI
−
<li>Remove blot from fridge.
+
s2uCEnOmhK0RsM64uH+q7BoUItalclULpGXVVevXrgWBznjZtGi1iRSrrLv8aXWUA0FzRu4abm2B
−
</li>
+
wL1i/73qFahNxbJ5357cWDeAxoC5Ixfkt/bzlmWP3+xb+j7YxS5FJx9yQAk1UWjsrfeQWFb1FpdJ
−
<li>Prepare primary antibody:
+
AOQE0XYDGtYSRGth2J9G0rnVH34T0YdahZkBNR4AXjG23kScUVhTK6cIpFyE4uHGImVzkZgXXGYB
−
<ul>
+
6FxZntnEIn7+ndTaU4K5pCNe7Q2iVEp1sffgeDrdJGWBDron4YNrLckWY57991ySF2JWk3JFeqYa
−
<li>10mls TBS</li>
+
aBp6OZZQKj55mXpUlufVTUCt91WCXA3UpE7GuRmWiGbpl6ZZOdrEn3PYYfRch4JKpWBWBCFImYEG
−
<li>2% Non-fat powdered milk</li>
+
GiURfBHl2VilYMoZKVVpCeejdQWZGhaSARLk3KhU7f/ZYEPldZdnQTiapCqsRH3lEC2mfrWrXnyl
−
<li>0.05% Tween</li>
+
Bp5ovFIV20NdwlbZa5MZlOdsyU41IqiDAEsga7g2Wq2y3tGaVSuQupWRrKlWSdi3U9FCC1QE+Rpa
−
</ul>
+
Ye/26CpiMbEbFnzZ+ZbisOAG9R1GX+n75mmBWZeoucI+F5/BDE7Y2In/FcaVp3YCDHFRjJqpmbyI
−
</li>
+
bryXxx7B5aBWr2n0W3Ai/aTxgFydtDBOT2n4sGsgX1uyryRWTFtXLx+knJ4uyyehXJR6xJXEBK0b
−
<li>
+
9FFOoVwQoUquy1CpiVhdqVjNFtQVkXuZtlJo1E4Vm6cmdY3iZMzhlNNGEqkdIWBP07Vp2alhqWRF
−
Add appropriate amount of antibody for dilution (1:5,000, 1:1,000, 1:500, etc.).
+
oS7/6HV6gPYklYigkZl1dk5a14rWB30dmtZpMV7UhovDRqTcYyfcZ2qXQ1d3SG87huNaUab4rEIF
−
</li>
+
xub1bQ5VHJt7mw3ikmo1n54aoBwthHFWGUWpXnCw49kxkcRd95TkfuKXqpr9gog8QgeCaBHIBdWL
−
<li>
+
akJodmw8o0XpjXRu4LMHcFeixntXv0o9Oe9ef87e722qtoc3bO37myX4jwVPEzb+Fc7qooqDXpCw
−
Prepare plastic bag:
+
NZqBaM8jVEtLdvxyrzZlTk1jss7/1PK5ikwrIheT1fcStJODeMV2c+uKmGJmOtx5RCfjGY9sAJSV
−
<ul>
+
mU1rV5zCHQdTxDvKzO8tajkONpgiweHBa2r2Qhyi/4anv4Uk8FA/LElgingqH75ENTJr21Hok5BN
−
<li>Cut plastic tube to size of gel.</li>
+
he9BrdCPAeHmMxR5Dz4mARoTF8I6qxwtKCpM1/nod7/v3XAxgugiXgCYFqWUC0UJM5XxanMc7YTk
−
<li>Seal bag with bag sealer set on 3.</li>
+
XXxMjWeSlMWpLWdh/evhzGCWm99h5hpzEpZBclUWaS2yI/rBBmFqxB6DNEmPJSRa7uTzH/69x4ld
−
<li>Put membrane blot in bag.</li>
+
fI60/MM1VtKKgVv04URc0aXnjUU2BvLeFYVjsk+pEXyqiwqOHlQlcaUoO6ELn56WpqvC8GUjpXKl
−
<li>Seal three sides of the plastic bag and cut excess (leave extra room on the top for when adding the
+
o5g2yaQ47JJ/AVLnfuVJzfkMlWBCCYScaR/7iXJCa4obCBGHQk7CrygTCyVWVAWsAhWwJKHbU+L4
−
antibody).</li>
+
JP+gEm3zPsKx3j1fWaQwFkWgRUqIAi0CTnwZrUGNLGbe5IK5pRHTZHHsJ54AEEcunqwzeZLi0YBZ
−
</ul>
+
tCX1sIexvF41IehMh3WmLzLZoYacFylanMudc/OdcCB5O/LwxJZUsdNkdgWsoFzsPRsZjxRhlcgL
−
</li>
+
Qa86gMHcUejZP+iBk11baqNE1bhBEWJkLMrJZ2+2Gi8woRFu2HTTh/7HRP55k5DQyVdU0mIzl3ow
−
<li>Add antibody with pipette and aide.</li>
+
nGoiK6zypNR1EmyGCZonqXLV1aQdxKw4qiApGaVFgjUUlppy1GOZOLxvJVN+DzRnGzslFktttjey
−
<li>Completely wet membrane.</li>
+
kdsw0cmrI/plh9ub4OU0uio6IjGLzbIPXhWbGcL/XvRo4vyiitKzo7tRMpS1yefu3gkAnuooZz/a
−
<li>Use a kimwipe to push out all the air bubbles.</li>
+
EFiEksCGuagtWdzeT4ZmnNQeEKiGyeoVkcaUXeWko/+h7aBkR7LlYcmnwBQvqbIHQefc0YLsIqE3
−
<li>Seal bag closed once all bubbles are removed and cut bag excess.</li>
+
f0SftIIrQsWxbYIepl59/RawYdTKOKVinMcisYCu8ILIECfZk/7TNMIySWNnkh7Efna7jdxSf7nj
−
<li>Put blot on rocker 1-2 hours at room temperature or overnight at 4 degrees Celsius.</li>
+
HYhK6cJZWmYx+8s/Nh34xN7UzoYzQ0LrovjAVgGJbz8s1JrpM4W5qlM73+glC2vQnX3lU1LlabrP
−
<li>Take blot out of the bag and put in TBST rinse.</li>
+
Ifc+zkWTi6+arCODc7qydbJqs3U1uKzsM0u5aYRW4rLt6knDhWSqWSflZQmNzYsd1m570irfHgri
−
<li>
+
/70UAVrtSsUSSTYZPw3DK2RLFt37VPEhfV3xRGUV3FQJ2pkLW3PGDn02oa7kOjCxiVtztmCFRDFA
−
TBST (For 1 Liter):
+
UqPwKZlnPiwexT3lrTSXwXPoR4tnwF9JkqhXzZBnbo5GPGG1rBey2+4qbNa4VigFhZvrXv/tTsrz
−
<ul>
+
tbCV+BQaDnvYOVGKaOB8bFHnEG7NZvXeOu24rvHFoNEWlFsD1LUwtadLZCsPj7PtmqXYsKetq86m
−
<li>900mls H2O</li>
+
sUZuEsk2rwi9LJmDU+p24+myXDwveOY7wQnZmztUI5Of8Su6Gdb7301xZ8ENdBEeIRHh3Immc6xm
−
<li>100mls 10X TBS</li>
+
YY/GGOKasaJWS7rAh2PcNWXij0QkFxsVGfXgHxGn8DmXgl2xpZw7/KurYrQWEAA7' />
−
<li>0.5mls Tween</li>
+
−
</ul>
+
−
</li>
+
−
 
+
−
<li>Save primary antibody. Store at 4 degrees Celsius.</li>
+
−
<li>Put blot in tray with 100-200mls TBST and put on rocker for 3-5 minutes.</li>
+
−
<li>Repeat rinse in TBST.</li>
+
−
<li>Prepare secondary antibody (1:5,000):
+
−
<ul>
+
−
<li>25mls TBST</li>
+
−
<li>5ul secondary antibody</li>
+
−
</ul>
+
−
</li>
+
−
<li> Pour secondary antibody onto blot and put on rocker for at least 1 hour.</li>
+
−
<li> Wash blot with 3 washes of TBST for 3-5 minutes each.</li>
+
−
<li> Prepare Western Blotting Detection Solution:
+
−
<ul>
+
−
 
+
−
<li>3mls solution A</li>
+
−
<li>3mls solution B</li>
+
−
</ul>
+
−
</li>
+
−
<li>Lay glass plate down on bench and lay blot on glass plate.</li>
+
−
<li>Put developer drop-wise over blots surface.</li>
+
−
<li>Tilt blot to make sure the whole blot is covered.</li>
+
−
<li>Let sit 1 minute.</li>
+
−
<li>Lay out a piece of smooth seran wrap.</li>
+
−
<li>Take blot off plate and let developer drip off.</li>
+
−
<li>Lay blot protein side down onto seran wrap and fold seran wrap to seal blot in bag.</li>
+
−
<li>Tape blot into cassette.</li>
+
−
<li>Take Biomax light film, cassette, timer and scissors down to the dark room.</li>
+
−
<li>Cut film into 2 pieces (fold upper right corner).</li>
+
−
<li>Put film over blot and close cassette.</li>
+
−
<li>Expose blot for 1 minute.</li>
+
−
<li>Put film in developer.</li>
+
−
<li>Expose blot for longer or shorter times depending on first exposure.</li>
+
−
<li>Label film using blot (when it is still taped into the cassette)</li>
+
−
</ol>
+
−
 
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
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−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group2">Coomassie Blue
+
−
Staining</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group2" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>II. Coomassie Blue Staining:</h1>
+
−
<h2>Materials and Reagents:</h2>
+
−
<h2>1. Coomassie Brilliant Blue R250 (EM Science) Equipment:</h2>
+
−
<ol>
+
−
<li>
+
−
Shaker
+
−
</li>
+
−
</ol>
+
−
<h2>
+
−
Procedure:
+
−
</h2>
+
−
<ol>
+
−
<li>Incubate the gel in staining solution with shaking for 30 min or longer (can leave it overnight).</li>
+
−
<li>Remove the dye solution (it can be reused for many times) and rinse the gel with water 1-2 times to remove
+
−
the dye.</li>
+
−
<li>Add destaining solution to the gel and incubate for 30-60 min.</li>
+
−
<li>Transfer the gel to water (can keep it in water for several days)</li>
+
−
</ol>
+
−
<h2>
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−
Recipes
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−
</h2>
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−
<ol>
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−
<li>
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−
100 ml Staining solution
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−
<ul>
+
−
<li>Coomassie Brilliant Blue R250 0.25 g</li>
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−
<li>Glacial acetic acid 10 ml</li>
+
−
<li>MetOH:H2O (1:1 v/v) 90 ml</li>
+
−
</ul>
+
−
</li>
+
−
<li>
+
−
Destaining solution
+
−
<p>
+
−
Destaining solution is the same as staining solution, but not containing the Coomassie R250 dye Powder.
+
−
</p>
+
−
</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
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−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group3">Silver Staining</a>
+
−
</h6>
+
−
</div>
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−
<div id="accordion-group3" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h2>III. Silver Staining:</h2>
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−
<ol>
+
−
<li>Fix gel in 10% MeOH / 10% Acetic Acid for 30 min or overnight</li>
+
−
<li>Wash 4X in H2O for at least 5 min</li>
+
−
<li>Incubate in sodium thiosulfate (1-2 pellets [400mg] per 500 ml) for 90 sec (Save 20 ml for later)</li>
+
−
<li>Wash 3X quickly with H2O</li>
+
−
<li>Add Silver nitrate solution (0.9 g silver nitrate in 500 mL H2O) for 10 min. Gel will turn slightly yellow</li>
+
−
<li>Wash the gel 3X quickly in H2O</li>
+
−
<li>Add developer solution (10g potassium carbonate, 20 ml sodium thiosulfate, 250 μl 40% Formaldehyde in 500 ml)</li>
+
−
<li>Stop the reaction by adding destain (10% MeOH and 5% Acetic Acid)</li>
+
−
<li>Wash gel in water</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
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<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group4">OVERLAP PCR</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group4" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h2>OVERLAP PCR:</h2>
+
−
<ol>
+
−
<li>order two primers which are complements of one another.</li>
+
−
<li>These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.</li>
+
−
<li>The "end primers" will not have any complements and will likely only have restriction sites.</li>
+
−
<li>PCR amplify the necessary fragments separately. Use a proofreading polymerase enzyme. Use an annealing temp
+
−
of 60°C.</li>
+
−
<li>Clean up the product using a DNA column.</li>
+
−
<li>"Overlap PCR" Use cleaned up fragments as template in a PCR reaction:</li>
+
−
<li>About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments. Do not
+
−
use Phusion polymerase. Try Pfu Turbo. Do not add any primers; the templates will prime each-other. 3.Run 15 PCR
+
−
cycles without primers.Use an annealing temp of 60°C.</li>
+
−
<li>Add end primers to the Overlap PCR reaction: Continue cycling for another 15-20 rounds. Use an annealing temp
+
−
of 72°C</li>
+
−
<li>Gel extract the correct size fragment.</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group5">Gel Extraction</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group5" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h2>V. Gel Extraction:</h2>
+
−
<h3>
+
−
Method:QIAquick Gel Extraction Kit I(250)
+
−
</h3>
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−
<ol>
+
−
<li>Excise the gel slice containing the DNA band with a clean, sharp scalpel. Minimize the size of the gel slice
+
−
by removing excess polyacrylamide.</li>
+
−
<li>Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel(i.e., 100–200 µl for each 100 mg
+
−
of gel).</li>
+
−
<li>Incubate at 50°C for 30 min.</li>
+
−
<li>Centrifuge the sample for 1 min.</li>
+
−
<li>Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet. Pass thesupernatant through a
+
−
disposable plastic column or a syringe containing either aWhatman GF/C filter or packed, siliconized glass wool
+
−
to remove any residualpolyacrylamide.</li>
+
−
<li>Determine the volume of the recovered supernatant.</li>
+
−
<li>Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the col or of the mixture is
+
−
yellow.</li>
+
−
<li>Place a QIAquick Spin Column in a provided 2 ml collection tube.</li>
+
−
<li>To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for30– 60 s.</li>
+
−
<li>Discard flow-through and place QIAquick Spin Column back into the same collection tube.</li>
+
−
<li>To wash, add 0.75 ml Buffer PE to column and centrifuge for 30–60 s.</li>
+
−
<li>Discard flow-through and place QIAquick Spin Column back in the same tube. Centrifuge column for an
+
−
additional
+
−
1 min at maximum speed.</li>
+
−
<li>Place QIAquick Spin Column into a clean 1.5 ml microcentrifuge tube.</li>
+
−
<li>To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of t he QIAquick Spin Column
+
−
and centrifuge for 1 min.</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group6">Plasmid Extraction</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group6" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h2>
+
−
VI. Plasmid Extraction:
+
−
</h2>
+
−
<h2>
+
−
Method:Plasmid Mini Kit I(200)
+
−
</h2>
+
−
<ol>
+
−
<li>Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium
+
−
containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (300
+
−
rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture.</li>
+
−
<li>Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.</li>
+
−
<li>Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell
+
−
pellet is vital for obtaining good yields.</li>
+
−
<li>Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.</li>
+
−
<li>Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed
+
−
to the next step.</li>
+
−
<li>Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.</li>
+
−
<li>Transfer the cleared supernatant from Step 5 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be
+
−
careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.</li>
+
−
<li>Centrifuge at 10000g speed for 1 minute.</li>
+
−
<li>Discard the filtrate and reuse the collection tube.</li>
+
−
<li>Add 500 µL HB Buffer.</li>
+
−
<li>Centrifuge at 10000g speed for 1 min.</li>
+
−
<li>Discard the filtrate and reuse collection tube.</li>
+
−
<li>Add 700 µL DNA Wash Buffer.</li>
+
−
<li>Centrifuge at 10000g for 1 minute.</li>
+
−
<li>Discard the filtrate and reuse the collection tube.</li>
+
−
<li>Centrifuge the empty HiBind® DNA Mini Column for2 min at 13000g to dry the column matrix.</li>
+
−
<li>Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.</li>
+
−
<li>Add 30-50 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.</li>
+
−
<li>Let sit at room temperature for 5 minute.</li>
+
−
<li>Centrifuge at 13000g for 1 minute.</li>
+
−
<li>Store DNA at -20°</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group7">Standard PCR</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group7" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h2>VII. Standard PCR:</h2>
+
−
<ol>
+
−
<li>Add DNA template up to 50ng into PCR tube,(x ul).</li>
+
−
<li>Add reagents as follow:
+
−
<ul>
+
−
<li>dNTP: 4ul</li>
+
−
<li>5x PSBuffer: 10ul</li>
+
−
<li>Primer: 1ul (after diluted)</li>
+
−
<li>Enzyme: 0.5ul</li>
+
−
<li>ddH2O: (33.5-x)ul</li>
+
−
</ul>
+
−
Total: 50ul<br />
+
−
Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
+
−
</li>
+
−
<li>
+
−
PCR procedure:<br />
+
−
98℃(2min)→ 98℃(10s)→55℃(5s)→72℃(1Kb/min)→72℃(5min)→4℃(∞)
+
−
</li>
+
−
<li>
+
−
The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary
+
−
to optimize the system for individual primers, template, and thermal cycler.
+
−
</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group8">PCR purification</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group8" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>
+
−
VIII. PCR purification：
+
−
</h1>
+
−
<h2>
+
−
Method:Cycle-Pure Kit(200)
+
−
</h2>
+
−
<ol>
+
−
<li>Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.</li>
+
−
<li>Determine the volume of the PCR reaction. Transfer the sample into a clean 1.5ml microcentrifuge tube and add
+
−
4-5 volumes of Buffer CP. For PCR products smaller than 200bp, add 6 volumes of Buffer CP.</li>
+
−
<li>Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the inside of the lid.</li>
+
−
<li>Place a HiBind DNA Mini Column into a provided 2 ml collection tube.</li>
+
−
<li>Add the mixed sample from step 3 to the HiBind DNA Mini Column and centrifuge at 13,000 x g for 1 minute at
+
−
room temperature. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same
+
−
collection tube.</li>
+
−
<li>Add 700µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and
+
−
place the HiBind DNA Mini Column back into the same collection tube.</li>
+
−
<li>Add 500µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and
+
−
place the HiBind DNA Mini Column back into the same collection tube.</li>
+
−
<li>Centrifuge the empty HiBind DNA Mini column for 2 min at maximal speed ( ≥13,000 x g ) to dry the column
+
−
matrix.</li>
+
−
<li>Place the HiBind DNA Mini column into a clean 1.5ml microcentrifuge tube. Depending on the desire
+
−
concentration of the final product, add 30-50 µl of Elution Buffer (10mM Tris, pH8.5) or water directly onto the
+
−
center of column matrix.</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group9">Colony PCR</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group9" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<ol>
+
−
<li>
+
−
Transform ligation mix or other plasmid-containing reaction mixture into the desired bacterial strain, and
+
−
incubate agar plates overnight at the appropriate temperature.
+
−
</li>
+
−
<li>
+
−
Set up 50 µl reactions as follows:
+
−
<table class="table table-bordered table-hover">
+
−
<tbody>
+
−
<tr>
+
−
<td>OneTaq Master Mix</td>
+
−
<td>25μl</td>
+
−
</tr>
+
−
<tr>
+
−
<td>PCR primer</td>
+
−
<td>
+
−
200 nM
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<td>H2O</td>
+
−
<td>to 50 µl</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
−
</li>
+
−
<li>
+
−
Use a sterile toothpick to pick up individual colonies and dip into each reaction tube.
+
−
</li>
+
−
<li>
+
−
As soon as the solution looks cloudy, remove the toothpick. To create a stock of each individual colony
+
−
either:
+
−
<p>
+
−
a.) Dip the toothpick into 3 ml growth media with appropriate antibiotics and culture overnight. or
+
−
</p>
+
−
<p>
+
−
b.) Streak the toothpick onto another agar plate containing the appropriate antibiotics and grow overnight.
+
−
</p>
+
−
</li>
+
−
<li>
+
−
Transfer reactions to a PCR cycler, and perform PCR following the guidelines below for cycling conditions:<br />
+
−
94℃(2min)→ 94℃(15s)→55℃(15s)→72℃(1Kb/min)→72℃(5min)→12℃(∞) </li>
+
−
<li>
+
−
Load 4-6 µl of each PCR reaction directly onto an agarose gel, alongside an appropriate DNA ladder.
+
−
</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group10">Agarose Gel
+
−
Electrophoresis</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group10" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>
+
−
X. Agarose Gel Electrophoresis：
+
−
</h1>
+
−
<ol>
+
−
<li>Weigh agarose powder and TAE buffer and add them to a flask;</li>
+
−
<li>Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave);</li>
+
−
<li>Let the solution cool to about 40-50℃ and pour the solution into the gel casting tray with appreciate comb;</li>
+
−
<li>Let the gel cool until it is solid;</li>
+
−
<li>Carefully pull out the comb; Place the gel in the electrophoresis chamber;</li>
+
−
<li>Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel;</li>
+
−
<li>Pipette DNA samples mixed with appreciate amount of loading buffer and dye (GeneFinder) into wells on the
+
−
gel;</li>
+
−
<li>Run the gel at 120V for about 20 minutes;</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group11">Extraction of E.
+
−
coli genomic DNA</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group11" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>XI. Extraction of E. coli genomic DNA:</h1>
+
−
<ol>
+
−
<li>E. coli shake flask culture one day in advance</li>
+
−
<li>1.5 ml of the inoculum was placed in a centrifuge tube, centrifuged at 5000 rpm for <br />1 min, and the
+
−
supernatant was discarded;</li>
+
−
<li>Add 190ul DD water suspension precipitation, and add 10ul 10% SDS, shake until the solution becomes viscous;</li>
+
−
<li>Add 1 ul of proteinase K (20 mg / ml), mix, incubate at 37 ° C for 1 hour;</li>
+
−
<li>Add 30ul 5mol / L NaCl, mix;</li>
+
−
<li>Add 30ul CTAB/NaCl solution, mix, heat at 65 ° C for 20min;</li>
+
−
<li>Add 300 ul of phenol/chloroform/isoamyl alcohol extraction (25:24:1), centrifuge at 5000 rpm for 10 min;</li>
+
−
<li>Take the supernatant, add 300 ul of chloroform / isoamyl alcohol (24:1), and centrifuge at 5000 rpm for 10
+
−
min;</li>
+
−
<li>Take the supernatant, add 300 ul of isopropanol, mix by inversion, stand at room temperature for 10 min,
+
−
precipitate the DNA, and centrifuge at 5000 rpm for 10 min;</li>
+
−
<li>Discard the supernatant, add 500ul 70% ethanol to wash the pellet, and centrifuge at 5000rmp for 10min;</li>
+
−
<li>Discard the supernatant and add 30 ul of DD water to dissolve the alcohol after it has evaporated.</li>
+
−
<li>Dissolve in 20 ul TE and store at -80 °C.</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group12">E.coli Competent
+
−
cells</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group12" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>
+
−
XII. E.coli Competent cells:
+
−
</h1>
+
−
<ol>
+
−
<li>Innoculate a single colony of E. coli into 5 ml LB and grow O/N at 37</li>
+
−
<li>Innoculate 1 ml into 100ml and grow to an O.D. 600 of 0.4</li>
+
−
<li>Aliquot the culture into 2x 50ml pre-chilled Sorvall tubes and leave on ice for 5 -10 mins</li>
+
−
<li>Centrifuge cells for 7 mins at 3000rpm, 4℃ without brakes</li>
+
−
<li>Pour off supernatant and resuspend each pellet in 10 ml of ice-cold CaCl2 soln</li>
+
−
<li>Centrifuge cells for 5 mins at 2500rpm, 4 degree. Discard supernatant and resuspend each pellet in 10 ml of
+
−
cold CaCl2 solution.</li>
+
−
<li>Keep resuspended cells on ice for 30min.</li>
+
−
<li>Centrifuge cells for 5 mins at 2500rpm, 4℃. Discard supernatant and resuspend each pellet in 2ml of ice-cold
+
−
CaCl2.</li>
+
−
<li>Dispense cells into pre-chilled sterile eppendorfs.</li>
+
−
<li>Freeze immediately @ -70 degresss</li>
+
−
</ol>
+
−
<p>
+
−
Notice: CaCl2 Soln: 60mM CaCl2, 15% Glycerol
+
−
</p>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group13">Protein Extraction
+
−
and expression</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group13" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
 
+
−
<h1>
+
−
XIII. Protein Extraction and expression:
+
−
</h1>
+
−
<ol type="a">
+
−
<li>
+
−
Bacterial transformation
+
−
<ol>
+
−
<li>80ul competent cells (BL21) + DNA samples (≤ 10ng; first add the plasmid and then add to small volume)</li>
+
−
<li>Place on ice for 30 min → 42 ° C for 45 s → place on ice for 1~2 min → add 1 ml of LB-Ep tube → shake at 37
+
−
° C for 1 h</li>
+
−
<li>Take 100ul on LB resistant plate, overnight at 37 ° C</li>
+
−
</ol>
+
−
</li>
+
−
<li>
+
−
IPTG induced expression
+
−
<ol>
+
−
<li>
+
−
Pick colonies on the plate and draw 8 (for example) small squares.
+
−
</li>
+
−
<li>
+
−
Pick up the bacteria with LB resistance solution, overnight at 37 ° C
+
−
(OD test method: (Cuvette in A312, tube photometer in A317) Click cell growth---general test---smart
+
−
test---cell growth (read-only) (wavelength is 600nm)---
+
−
measure Blanket (what is used as a blank for dilution, such a test should be blank with LB)---measure sample)
+
−
</li>
+
−
<li>
+
−
Cuvette liquid OD value, OD = A, diluted into Bml
+
−
<p>
+
−
(B * 0.2 / A = C, Bml LBK added Cml bacteria), OD = 0.2 (recommended to be diluted to 5ml or 10ml at the
+
−
beginning )
+
−
Shake for 2h at 37℃, generally to OD = 0.5;
+
−
</p>
+
−
</li>
+
−
<li>Add IPTG induction generally optimally 0.5mM</li>
+
−
<li>Initially induced expression requires exploration conditions: 1 IPTG amount: 0.1 ~ 1 mM gradient (0, 0.25,
+
−
0.5, 1)</li>
+
−
<li>4 hours(depend on the type of the protein) of culture at 37 degrees</li>
+
−
<li>Centrifuge the cells to precipitate and wash them twice with PBS (make sure to wash) (add 3ml PBS) (move
+
−
into the Ep tube when washing with PBS for the first time)</li>
+
−
</ol>
+
−
</li>
+
−
<li>
+
−
Protein extraction
+
−
<ol>
+
−
<li>
+
−
lysate with mix: PMSF (1:40). protein inhibitor (1:100), DTT (500 mM → 5 mM); Add ulsate 500ul per tube;
+
−
Ultrasonic disruption.
+
−
</li>
+
−
<li>
+
−
Centrifuge at 44 ° C, 13000 rpm for 20 min, take the supernatant
+
−
</li>
+
−
</ol>
+
−
</li>
+
−
</ol>
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
<div class="panel panel-white">
+
−
<div class="panel-heading">
+
−
<h6 class="panel-title">
+
−
<a class="collapsed" data-toggle="collapse" data-parent="#accordion1" href="#accordion-group14">Protein Extraction</a>
+
−
</h6>
+
−
</div>
+
−
<div id="accordion-group14" class="panel-collapse collapse">
+
−
<div class="panel-body">
+
−
<h1>
+
−
XIV. List of some kits and reagents
+
−
</h1>
+
−
 
+
−
 
+
−
<table class="table table-hover table-bordered table-striped">
+
−
<tbody>
+
−
<tr>
+
−
<th colspan="2">List of experimental kits</th>
+
−
<th colspan="2">List of biological reagents</th>
+
−
</tr>
+
−
<tr>
+
−
<td>kits </td>
+
−
<td>origin </td>
+
−
<td>anti-Ha, anti-Flag antibody </td>
+
−
<td>Sigma,Abcam</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Plasmid Mini Kit I(200)</td>
+
−
<td>Omega </td>
+
−
<td>anti-rabbit IgG </td>
+
−
<td>Proteintech</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Plasmid Mini Kit(25)</td>
+
−
<td>Omega</td>
+
−
<td>anti-mouse IgG </td>
+
−
<td>Proteintech</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Cycle-Pure Kit(200) </td>
+
−
<td>Omega </td>
+
−
<td>GoTaq Colonrless Master </td>
+
−
<td>Promega</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Gel Extraction Kit I(200)</td>
+
−
<td>Omega </td>
+
−
<td>PrimerSTAR Max DNA Polymerase</td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Endo-free Plasmid Mini Kit II(50)</td>
+
−
<td>Omega </td>
+
−
<td>PrimerSTAR HS DNS Polymerase</td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>GoScript Reverse Transcription System </td>
+
−
<td>Promega </td>
+
−
<td>DL2000 DNA Marker </td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>QIAquick PCR Purification Kit(250)</td>
+
−
<td>QIAGEN </td>
+
−
<td>1Kb DNA Ladder(Dye Plus) </td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>QIAquick Gel Extraction Kit I(250)</td>
+
−
<td>QIAGEN </td>
+
−
<td>6x DNA Loading Buffer </td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>In-Fusion HD Cloning Kit </td>
+
−
<td>Takara </td>
+
−
<td>10x DNA Loading Buffer </td>
+
−
<td>Takara</td>
+
−
</tr>
+
−
<tr>
+
−
<td>DNA Ligation Kit Ver.2.1</td>
+
−
<td>Takara </td>
+
−
<td>Protease Inhibitor Cocktail </td>
+
−
<td>CWBIO</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Blunting Kination Ligation Kit</td>
+
−
<td>Takara </td>
+
−
<td>2x Es Taq Master Mix </td>
+
−
<td>CWBIO</td>
+
−
</tr>
+
−
<tr>
+
−
<td>BCA Protein Assay Kit </td>
+
−
<td>CWBIO </td>
+
−
<td>Protease Inhibitor 8340 </td>
+
−
<td>Sigma</td>
+
−
</tr>
+
−
<tr>
+
−
<td>cECL Western Blot Kit </td>
+
−
<td>CWBIO </td>
+
−
<td>PMSF </td>
+
−
<td>Sigma</td>
+
−
</tr>
+
−
<tr>
+
−
<td>ECL Western Blot Kit </td>
+
−
<td>WBIO </td>
+
−
<td>restained Protein Ladder </td>
+
−
<td>hermo</td>
+
−
</tr>
+
−
<tr>
+
−
<td>Plant RNA kit </td>
+
−
<td>OMEGA </td>
+
−
<td>Xma I, Restriction enzyme </td>
+
−
<td>NEB</td>
+
−
</tr>
+
−
<tr>
+
−
<td>&nbsp;</td>
+
−
<td>&nbsp;</td>
+
−
<td>
+
−
Apa I, BamH I, EcoR I, , Kpn
+
−
I, Not I, Sal I, Xba I, Spe I,
+
−
Pst I, Xba I
+
−
</td>
+
−
<td>
+
−
Takara
+
−
</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
−
 
+
−
</div>
+
−
</div>
+
−
</div>
+
−
 
+
−
</div>
+

Revision as of 02:24, 15 October 2018

Ruby - Responsive Corporate Tempalte

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