Revision as of 13:24, 4 October 2016

TEAM TIANJIN

Team Tianjin-Attribution

Protocol

Plasmid Extraction

We use the TIANprep Mini Plasmid Kit made by TIANGEN Biotech Co.,Ltd. to extract plasmid. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.
1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
2. Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
3. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
Note: No cell clumps should be visible after resuspension ofthe pellet, otherwise incomplete lysis will lower yield and purity. 4. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.
Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
5. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. (Optional, actually we hardly ever use) Wash the Spin Column CP3 by adding 500 μl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube.
This step is recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
8. Wash the Spin Column CP3 by adding 600 μl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
9. Repeat Step 8.
10. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
11. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.

DNA Purification

We use the TIANquick Midi Purification Kit made by TIANGEN Biotech Co.,Ltd. to purify the DNA products of PCR and restriction endonuclease cutting. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).
1. Column equilibration: add 500 μl Buffer BL to the Spin Column CB2 (put Spin Column CB2 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g). Discard the flow-through, and then place Spin Column CB2 back into the collection tube (please use freshly treated spin column).
2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.
Note: For example, add 250 μl Buffer PB to 50 μl PCR reaction (not including oil).
3. Transfer the mixture to the Spin Column CB2, incubate at room temperature (15-25°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB2 back into the same collection tube.
Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.
4. Add 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB2 and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB2 back in the same collection tube.
Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.
5. Repeat step 4.
6. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.
Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).
7. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. Add 30-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 30 μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.

Agarose Gel Electrophoresis Products Recycling

We use the TIANgel Midi DNA Purification Kit made by TIANGEN Biotech Co.,Ltd. to recycle the DNA products from the agarose gel. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).
1. Column equilibration: add 500 μl Buffer BL to the Spin Column CA2 (put Spin Column CA2into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put Spin Column CA2 back into the collection tube (please use freshly treated spin column).
2. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean tube.
3. Add equivalent volume of Buffer PN to the gel (If the gel is 0.1 g, it is defaulted to be 100 μl, then add 100 μl Buffer PN). Incubate at 50°C by inverting up and down the tube until the agarose gel dissolves completely. If the agarose gel does not dissolve completely, incubate for longer period or add additional Buffer PN until all the agarose gel dissolved completely (If the agarose gel is too large, please cut the agarose gel into several pieces in advance).
Note: If DNA fragment is <300 bp, it is recommended to add isopropanol which is 1/2 volume of Buffer PN to the agarose gel sample after the gel completely dissolved. Cooling the solution at room temperature (15-25°C) and then add the solution to Spin Column CA2 since silica membrane of the column adsorbs DNA best at room temperature.
4. When the gel dissolved completely and the solution temperature turns to room temperature (15-25°C), transfer the mixture to the Spin Column CA2 (put Spin Column CA2into a collection tube). Let the column stand for 2 min at room temperature (15-25°C), then centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through; place the Spin Column CA2 back into the collection tube again.
Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.
5. Wash the Spin Column CA2 with 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and place the Spin Column CA2 back into the collection tube.
Note: If the purified DNA is used for the salt sensitive experiments, such as direct sequencing and blunt-ended ligation, let the column stand for 2-5 min after adding Buffer PW, and then centrifuge.
6. Repeat Step 5.
7. Place the Spin Column CA2 back to the collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual wash buffer. Discard the flow-through, and place column with the cap open for several minutes to air dry the membrane.
Note: Residual ethanol from Buffer PW will influence the subsequent enzymatic reaction (enzyme digestion, PCR etc).
8. Transfer the Spin Column CA2 to a clean 1.5 ml microcentrifuge tube. Add appropriate volume of Buffer EB to the center of the membrane, incubate at room temperature (15-25°C) for 2 min, then centrifuge at 12,000 rpm (~13,400 × g) for 2 min.
Note: The elution volume should not be less than 30 μl since smaller volume will affect recovery efficiency. The pH value of eluted buffer will affect eluting. If purified DNA is used for sequencing, it is recommended to choose ddH2O (pH 7.0-8.5) to elute DNA, pH<7.0 will decrease the elution efficiency. Obtained DNA should be stored at -20°C to prevent degradation. Buffer (10 mM Tris-Cl, pH 8.0) could also be used for DNA elution. For higher yield, pipette the eluate to the center of the membrane again, incubate 2 min and centrifuge at 12,000 rpm (~13,400 × g) for 2 min.

Agarose Gel Electrophoresis

1. Preparation of TAE buffer: Add 242g Tris, 37.2g Na2EDTA·2H2O, 800mL ddH2O, after all the solute dissolve, add 57.1mL acetic acid and add ddH2O to make the total volume 1L.
2. Preparation of sample: Add DNA loading buffer to the DNA solution according to the dilution ratio of particular buffer.
3. Preparation of agarose gel: Add agarose powder 10g/L, 1×TAE buffer 100mL (variable, according to need), heat the mixture by microwave oven until the agarose was dissolved. After the solution cool down to touchable temperature, add 50-100μL/L Goldenview Nucleic acid dye to the solution. Then pour the solution to the gel mould with gel comb inserted and wait for its concretion.
4. Add samples to the gel pore: After the formation of gel, pull out the gel comb and take the gel out of the mould. Immerge the gel with 1×TAE buffer in the electrophoresis chamber. Using pipette to add marker and samples to different pore. (The content of pore depends on the gel comb, there are 3 kinds of volume, 10μL, 20μL, and 50μL) Do not stick the bottom and side of gel pore to prevent the leakage.
5. Turn on the electrical source to start the electrophoresis, the voltage is set at 150-160V and the electrophoresis time is set at 8-12min.
6. After the electrophoresis process end, the gel is observed under blue light or ultraviolet.

Restriction Endonuclease Digestion

Prepare the system:

Total volume: 50μL

Restriction endonuclease: 2μL respectively

10×Cut Smart Buffer: 5μL

DNA to be cut: 30μL

ddH2O: 13μL

Reaction time: 2h

Reaction temperature: 37℃

DNA Fragments Ligation

Prepare the system:

Total volume: 20μL

T4 DNA ligase: 1μL

5×Ligase Buffer: 4μL

DNA to be linked: (c1V1/L1): (c2V2/L2)=5:1. (c1: Concentration of cut DNA fragments; c2: Concentration of cut plasmid; V1: Volume of cut DNA fragments; V2: Volume of cut plasmid; L1: Length of cut DNA fragments; L2: Length of cut plasmid)

ddH2O: add to make the total volume 20μL

Reaction time: 2h

Reaction temperature: 22℃

PCR

Prepare the system:

Total volume: 50μL

Q5 DNA Polymerase: 0.5μL

5×Q5 DNA Polymerase Buffer: 10μL

Template: 1μL

dNTP: 1μL

Primer: Sense Primer and Anti-sense Primer, respectively 2.5μL

ddH2O: 32.5μL

Cycles: 25-35

Pre-denaturation: 98℃,30s;

Denaturation: 98℃,5-10s;

Annealing: Depend on the primers, generally 45-65℃,10-30s;

Extension: 72℃,20-30s/kb

Fully extension: 72℃,2min

Product Storage: 4℃

Note: Different DNA polymerase has different protocol, this is only the case of Q5 DNA polymerase.

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Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin

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         <div id="content" class="home blog single-author one-column content" role="main">
-<!--------------------------------week1 start-------------------------------------------->
   <div id="Week1"></div>
 <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 	<header class="entry-header">
-		<div class="entry-title" align="center" >Notes For R-R system</div>
+		<div class="entry-title" align="center" >Protocol</div>
 	</header><!-- .entry-header -->
-         <h1 class="entry-title">Week1(8/24/2016-8/30/2016)</h1>
+         <h1 class="entry-title">Plasmid Extraction</h1>
 		<div class="entry-content">
                    <div class="note-content">
-    <li>We used PCR to amplify the <b><i>CpxR</i></b> promoter and <b><i>RFP</i></b> gene from plasmid <b><i>pUC57</i></b>, and then we recycled the amplified fragment from the agarose gel. Then we used<b><i> Xba1</i></b> and <b><i>Pst1</i></b> enzymes to cut the plasmid <b><i>pUC19</i></b> and <b><i>CpxR-RFP</i></b> fragment. </li>
+We use the TIANprep Mini Plasmid Kit made by TIANGEN Biotech Co.,Ltd. to extract plasmid. Here is the protocol.<br/>
-   <li>We linked the cut plasmid and<b><i> CpxR-RFP</i></b> fragment together and transformed the recombinant plasmid to <b><i>E.coli</i></b>.
+Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.<br/>
-We used PCR to amplify the <b><i>PETase</i></b> gene and then recycled them from the agarose gel.</li>
+. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).<br/>
-<li>We cultured the grown-up<b><i> E.coli</i></b> which had been transformed into recombinant <b><i>pUC19</i></b> into liquid LB+Amp culture medium overnight.</li>
+. Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)<br/>
-<li>We isolated the recombinant plasmid from the<b><i> E.coli</i></b> cultured last night. Then we use<b><i> Xba1</i></b> and <b><i>Pst1</i></b> enzyme to cut the plasmid to verify the plasmid was successfully constructed. The result was we succeeded.</li>
+. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.<br/>
-<li>We cut the recombinant plasmid <b><i>pUC19</i></b> with enzyme<b><i> EcoR1</i></b> and <b><i>Sac1</i></b> and then we recycled it from the agarose gel. We stored the recycled product in -30℃ in order to wait for the <b><i>PETase</i></b> gene transformed into it.</li>
+Note: No cell clumps should be visible after resuspension ofthe pellet, otherwise incomplete lysis will lower yield and purity.
-<li>In order to verify the inclusion body sensing effects of <b><i>CpxR</i></b> promoter, we selected a colony of <b><i>E.coli</i></b> with recombinant plasmid<b><i> pUC19</i></b> and cultured them in 37℃ for 6 hours and then rose the temperature to 42℃ and culture it overnight.</li>
+. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.<br/>
-<li>The result of the verification experiment last night was unsuccessful for there was only ultralow red fluorescence was detected, which was considered the basic expression of <b><i>RFP</i></b>.</li>
+Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.<br/>
-<li>We redesigned the experiment and set 3 groups:<br/>
+. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.<br/>
-. E.coli with standard <b><i>RFP</i></b> gene from our own laboratory.<br/>
+Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.<br/>
-. E.coli with our recombinant plasmid<b><i> pUC19</i></b> and we cultured them in 37℃ all through.<br/>
+. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.<br/>
-. E.coli with our recombinant plasmid <b><i>pUC19</i></b> and we first cultured them in 37℃ and after 6 hours transferred them to 42℃.
+. (Optional, actually we hardly ever use) Wash the Spin Column CP3 by adding 500 μl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube.<br/>
-</li>
+This step is recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.<br/>
-<li>The result was still unsuccessful for the 2nd and 3rd group showed ultralow red fluorescence and only 1st group showed high enough red fluorescence.</li>
+. Wash the Spin Column CP3 by adding 600 μl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.<br/>
-<li>We redesigned the experiment again. We decided to transformed the recombinant plasmid <b><i>pUC19</i></b> and<b><i> pET21a</i></b> which was from the protein modification group and had <b><i>PETase</i></b> gene in it into<b><i> E.coli</i></b> at the same time.</li>
+. Repeat Step 8.<br/>
-<li>We cut the<b><i> pUC57</i></b> with enzyme <b><i>Xba1</i></b> and <b><i>Pst1</i></b> and recycled the skeleton part from the agarose gel.</li>
+. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.<br/>
+Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.<br/>
+. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).<br/>
+Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.
 </div>
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 	     </article> #post-4252 -->
-<!------------------------------------week1 end------------------------------------------------>
-<!------------------------------------week2 start------------------------------------------------>
 <div id="Week2"></div>
 <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 	<header class="entry-header">
-		 <h1 class="entry-title">Week2(8/31/2016-9/6/2016)</h1>
+		 <h1 class="entry-title">DNA Purification</h1>
 	</header><!-- .entry-header -->
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 <div class="note-content2">
-<li>We linked the remained cut <b><i>CpxR-RFP</i></b> fragment into the skeleton and then transformed the recombinant <b><i>pUC57</i></b> and the <b><i>pET21a</i></b> into<b><i> E.coli</i></b> at the same time.</li>
+We use the TIANquick Midi Purification Kit made by TIANGEN Biotech Co.,Ltd. to purify the DNA products of PCR and restriction endonuclease cutting. Here is the protocol.<br/>
+Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).<br/>
+. Column equilibration: add 500 μl Buffer BL to the Spin Column CB2 (put Spin Column CB2 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g). Discard the flow-through, and then place Spin Column CB2 back into the collection tube (please use freshly treated spin column).<br/>
+. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.<br/>
+Note: For example, add 250 μl Buffer PB to 50 μl PCR reaction (not including oil).<br/>
+. Transfer the mixture to the Spin Column CB2, incubate at room temperature (15-25°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB2 back into the same collection tube.<br/>
+Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.<br/>
+. Add 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB2 and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB2 back in the same collection tube.<br/>
+Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.<br/>
+. Repeat step 4.<br/>
+. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.<br/>
+Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).<br/>
+. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. Add 30-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).<br/>
+Note: If the volume of eluted buffer is less than 30 μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.
-<li>The transformation last night turned to be a failure. We tried it again.</li>
-<li>The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.</li>
-<li>We finally gave up the former design and decided to link the<b><i> PETase</i></b> gene into the plasmid<b><i> pUC19</i></b>. However, we did not have the key enzyme <b><i>Sal1</i></b> so we started to construct the TPA positive feedback system.</li>
-<li>We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the <b><i>TPA-sensing leader sequence</i></b>,<b><i> PGK1 promoter</i></b>, <b><i>CYC1 terminator</i></b>, <b><i>RFP gene</i></b>, TPA regulation protein gene (<b><i>tpaR</i></b>), TPA transporting protein gene (<b><i>tpaK</i></b>). Then we cut the fragments above and plasmid <b><i>pRS413</i></b>, <b><i>pRS415</i></b>, and <b><i>pYES2</i></b> with corresponding enzymes and recycled the fragments from agarose gel.</li>
-<li>We linked the fragments together by this way:<br/>
-<b><i>1. pYES2-leader-PGK1-RFP.<br/>
-. pRS413-PGK1-tpaK-CYC1.<br/>
-. pRS415-PGK1-tpaR-CYC1</i></b>
-</li>
-<li>Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into <b><i>Saccharomyces cerevisiae</i></b>.
-</li>
-<li>The key enzyme <b><i>Sal1</i></b> arrived and we isolate the plasmid <b><i>pET21a</i></b>. Then we use <b><i>BamH1</i></b> and <b><i>Sal1</i></b> to cut both plasmid and <b><i>PETase</i></b> gene, then linked them together and transformed the recombinant plasmid into <b><i>E.coli</i></b>.</li>
 </div>
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-<!------------------------------------week2 end------------------------------------------------>
-<!------------------------------------week3 start------------------------------------------------>
 <div id="Week3"></div>
 <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-	<h1 class="entry-title">Week3(9/7/2016-9/13/2016)</h1>
+	<h1 class="entry-title">Agarose Gel Electrophoresis Products Recycling</h1>
 		<div class="entry-content">
@@ Line 154: / Line 152: @@
-<li>We cultured the transformed<b><i> E.coli</i></b> and isolated the plasmid. Then we use PCR to amplify the whole fragment in <b><i>pET21a</i></b> from <b><i>T7 promoter</i></b> to <b><i>T7 terminator</i></b>. Then we recycled this fragment from agarose gel.</li>
+We use the TIANgel Midi DNA Purification Kit made by TIANGEN Biotech Co.,Ltd. to recycle the DNA products from the agarose gel. Here is the protocol.<br/>
-<li>The transformed <b><i>Saccharomyces cerevisiae</i></b> had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.</li>
+Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).<br/>
-<li>We cut the <b><i>T7 promoter-PETase gene-T7 terminator</i></b> fragment with enzymes <b><i>EcoR1</i></b> and <b><i>Sac1</i></b>. Then we linked it to the already cut plasmid <b><i>pUC19</i></b> (cut in August 28th). Then we transformed the recombinant plasmid into<b><i> E.coli</i></b>.</li>
+. Column equilibration: add 500 μl Buffer BL to the Spin Column CA2 (put Spin Column CA2into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put Spin Column CA2 back into the collection tube (please use freshly treated spin column).<br/>
-<li>We cultured the transformed <b><i>Saccharomyces cerevisiae</i></b> into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:<br/>
+. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean tube. <br/>
-. Group 1: did not add TPA.<br/>
+. Add equivalent volume of Buffer PN to the gel (If the gel is 0.1 g, it is defaulted to be 100 μl, then add 100 μl Buffer PN). Incubate at 50°C by inverting up and down the tube until the agarose gel dissolves completely. If the agarose gel does not dissolve completely, incubate for longer period or add additional Buffer PN until all the agarose gel dissolved completely (If the agarose gel is too large, please cut the agarose gel into several pieces in advance). <br/>
-. Group 2: added 1000μL TPA standard solution.<br/>
+Note: If DNA fragment is <300 bp, it is recommended to add isopropanol which is 1/2 volume of Buffer PN to the agarose gel sample after the gel completely dissolved. Cooling the solution at room temperature (15-25°C) and then add the solution to Spin Column CA2 since silica membrane of the column adsorbs DNA best at room temperature. <br/>
-. Group 3: added 100μL TPA standard solution.<br/>
+. When the gel dissolved completely and the solution temperature turns to room temperature (15-25°C), transfer the mixture to the Spin Column CA2 (put Spin Column CA2into a collection tube). Let the column stand for 2 min at room temperature (15-25°C), then centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through; place the Spin Column CA2 back into the collection tube again. <br/>
-. Group 4: added 10μL TPA standard solution.<br/>
+Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.<br/>
-.Group 5: added 1μL TPA standard solution.
+. Wash the Spin Column CA2 with 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and place the Spin Column CA2 back into the collection tube. <br/>
-</li>
+Note: If the purified DNA is used for the salt sensitive experiments, such as direct sequencing and blunt-ended ligation, let the column stand for 2-5 min after adding Buffer PW, and then centrifuge. <br/>
-<li>We cultured the transformed <b><i>E.coli</i></b> into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of <b><i>PETase</i></b> gene.</li>
+. Repeat Step 5. <br/>
-<li>We first detected the red fluorescence of <b><i>E.coli</i></b>, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.</li>
+. Place the Spin Column CA2 back to the collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual wash buffer. Discard the flow-through, and place column with the cap open for several minutes to air dry the membrane. <br/>
-<li>The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.</li>
+Note: Residual ethanol from Buffer PW will influence the subsequent enzymatic reaction (enzyme digestion, PCR etc). <br/>
-<li>We doubted that it might be the<b><i> RFP</i></b> in the kit was useless. We isolated the <b><i>pET21a</i></b> and used PCR to amplify the <b><i>RFP </i></b>gene.</li>
+. Transfer the Spin Column CA2 to a clean 1.5 ml microcentrifuge tube. Add appropriate volume of Buffer EB to the center of the membrane, incubate at room temperature (15-25°C) for 2 min, then centrifuge at 12,000 rpm (~13,400 × g) for 2 min. <br/>
-<li>We cut the <b><i>RFP</i></b> gene and <b><i>pET21a</i></b> with enzymes <b><i>Xba1</i></b> and <b><i>Sac1</i></b>, then we linked them and transformed it into <b><i>E.coli</i></b>.</li>
+Note: The elution volume should not be less than 30 μl since smaller volume will affect recovery efficiency. The pH value of eluted buffer will affect eluting. If purified DNA is used for sequencing, it is recommended to choose ddH2O (pH 7.0-8.5) to elute DNA,    pH<7.0 will decrease the elution efficiency. Obtained DNA should be stored at -20°C to prevent degradation. Buffer (10 mM Tris-Cl, pH 8.0) could also be used for DNA elution. For higher yield, pipette the eluate to the center of the membrane again, incubate 2 min and centrifuge at 12,000 rpm (~13,400 × g) for 2 min.
-<li>We cultured the transformed <b><i>E.coli</i></b> and added IPTG to induce the expression of<b><i> RFP</i></b>, and this time the red fluorescence was clear enough that could be seen directly.</li>
 </div>
@@ Line 186: / Line 183: @@
-<!------------------------------------week3 end------------------------------------------------>
-<!------------------------------------week4 start------------------------------------------------>
 <div id="Week4"></div>
 <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-	<h1 class="entry-title">Week4(9/14/2016-9/20/2016)</h1>
+	<h1 class="entry-title">Agarose Gel Electrophoresis</h1>
 		<div class="entry-content">
@@ Line 201: / Line 193: @@
 <div class="note-content4">
-<li>We started to construct another regulation way, the <b><i>E.coli</i></b> lysis regulation pathway. We first used colony PCR to obtain the <b><i>ddpX</i></b> gene from the <b><i>E.coli</i></b> genome and recycled the <b><i>ddpX</i></b> from the agarose gel.</li>
+. Preparation of TAE buffer: Add 242g Tris, 37.2g Na2EDTA·2H2O, 800mL ddH2O, after all the solute dissolve, add 57.1mL acetic acid and add ddH2O to make the total volume 1L.<br/>
+. Preparation of sample: Add DNA loading buffer to the DNA solution according to the dilution ratio of particular buffer.<br/>
+. Preparation of agarose gel: Add agarose powder 10g/L, 1×TAE buffer 100mL (variable, according to need), heat the mixture by microwave oven until the agarose was dissolved. After the solution cool down to touchable temperature, add 50-100μL/L Goldenview Nucleic acid dye to the solution. Then pour the solution to the gel mould with gel comb inserted and wait for its concretion.<br/>
+. Add samples to the gel pore: After the formation of gel, pull out the gel comb and take the gel out of the mould. Immerge the gel with 1×TAE buffer in the electrophoresis chamber. Using pipette to add marker and samples to different pore. (The content of pore depends on the gel comb, there are 3 kinds of volume, 10μL, 20μL, and 50μL) Do not stick the bottom and side of gel pore to prevent the leakage. <br/>
+. Turn on the electrical source to start the electrophoresis, the voltage is set at 150-160V and the electrophoresis time is set at 8-12min.<br/>
+. After the electrophoresis process end, the gel is observed under blue light or ultraviolet.
-<li>We found that there was no enzyme cleavage site between the<b><i> CpxR</i></b> promoter and <b><i>RFP</i></b> gene in the part we use. We had to design the primers and amplified the <b><i>CpxR</i></b> promoter by PCR.</li>
-<li>We used PCR to amplify the<b><i> CpxR</i></b> promoter. Then we recycled it from agarose gel.</li>
-<li>We cut the<b><i> CpxR</i></b> promoter with enzymes <b><i>Xba1</i></b> and<b><i> BamH1</i></b>, <b><i>ddpX</i></b> gene with enzymes <b><i>BamH1</i></b> and<b><i> EcoR1</i></b>, first batch of <b><i>pET21a</i></b> with<b><i> Xba1</i></b> and<b><i> EcoR1</i></b>, second batch of <b><i>pET21a</i></b> with<b><i> BamH1</i></b> and <b><i>EcoR1</i></b>.</li>
-<li>Then we linked these fragment in the following two ways:<br/>
-<b><i>1. pET21a-CpxR-ddpX.<br/>
-. pET21a-ddpX.</i></b>
-</li>
@@ Line 224: / Line 214: @@
-<!------------------------------------week4 end------------------------------------------------>
-<!------------------------------------week5 start------------------------------------------------>
 <div id="Week5"></div>
 <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-	<h1 class="entry-title">Week5(9/21/2016-9/27/2016)</h1>
+	<h1 class="entry-title">Restriction Endonuclease Digestion</h1>
 		<div class="entry-content">
@@ Line 238: / Line 222: @@
 <div class="note-content5">
-<li>We used PCR to amplify the whole fragments in<b><i> pET21a</i></b> (from <b><i>CpxR </i></b>to <b><i>T7 terminator</i></b>). However, the band in the agarose gel was disperse so that we were unable to recycle it.</li>
+Prepare the system:
+<li>Total volume: 50μL</li>
-<li>We used colony PCR to verify if the <b><i>pET21a</i></b> had been correctly constructed, the result is yes.</li>
+<li>Restriction endonuclease: 2μL respectively</li>
-<li>We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.</li>
+<li>10×Cut Smart Buffer: 5μL</li>
-<li>We cultured the <b><i>E.coli</i></b> transformed into the <b><i>pET21a-ddpX</i></b> fragment and detect the OD600 in order to verify the lysis effection of <b><i>ddpX</i></b>. </li>
+<li>DNA to be cut: 30μL</li>
-<li>Considering the <b><i>pYES2</i></b> is multicopy plasmid so that the copy number would affect the <b><i>RFP</i></b> expression level, we decided to change the <b><i>pYES2</i></b> to single-copy plasmid <b><i>pRS416</i></b>. Since the <b><i>pRS416</i></b> does not have terminator in its backbone, we used PCR to amplify the <b><i>CYC1</i></b> terminator from plasmid <b><i>pYES2</i></b>.</li>
+<li>ddH2O: 13μL</li>
-<li>We cut the <b><i>pYES2</i></b> with enzyme <b><i>Hind3</i></b> and <b><i>EcoR1</i></b>, <b><i>CYC1</i></b> with<b><i> EcoR1</i></b> and <b><i>Sal1</i></b>,<b><i> pRS416</i></b> with <b><i>Hind3</i></b> and<b><i> Sal1</i></b>. Then we linked the three part together.</li>
+<br/>
-<li>We transformed the three plasmids into <b><i>Saccharomyces cerevisiae</i></b> together. </li>
+<li>Reaction time: 2h</li>
-<li>The new primers using to amplify the<b><i> CpxR-ddpX-T7</i></b> terminator fragment arrived and we redid the PCR. However, the disperse band was still existed. </li>
+<li>Reaction temperature: 37℃</li>
-<li>The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate. </li>
@@ Line 265: / Line 248: @@
-<!------------------------------------week5 end------------------------------------------------>
-<!------------------------------------week6 start------------------------------------------------>
   <div id="Week6"></div>
          <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 	<header class="entry-header">
-		<h1 class="entry-title">Week6(9/28/2016-10/2/2016)</h1>
+		<h1 class="entry-title">DNA Fragments Ligation</h1>
 	</header><!-- .entry-header -->
@@ Line 281: / Line 260: @@
 <div class="note-content6">
-<li>We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with <b><i>PETase</i></b> gene and <b><i>CpxR-RFP</i></b> fragment showed the deepest red.</li>
+Prepare the system:
+<li>Total volume: 20μL</li>
+<li>T4 DNA ligase: 1μL </li>
+<li>5×Ligase Buffer: 4μL</li>
+<li>DNA to be linked: (c1V1/L1): (c2V2/L2)=5:1. (c1: Concentration of cut DNA fragments; c2: Concentration of cut plasmid; V1: Volume of cut DNA fragments; V2: Volume of cut plasmid; L1: Length of cut DNA fragments; L2: Length of cut plasmid)</li>
+<li>ddH2O: add to make the total volume 20μL</li>
+<br/>
+<li>Reaction time: 2h</li>
+<li>Reaction temperature: 22℃</li>
@@ Line 290: / Line 278: @@
 		</div><!-- .entry-content -->
+<div id="Week7"></div>
+        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">PCR</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+<div class="note-content7">
+Prepare the system:
+<li>Total volume: 50μL</li>
+<li>Q5 DNA Polymerase: 0.5μL</li>
+<li>5×Q5 DNA Polymerase Buffer: 10μL</li>
+<li>Template: 1μL</li>
+<li>dNTP: 1μL</li>
+<li>Primer: Sense Primer and Anti-sense Primer, respectively 2.5μL</li>
+<li>ddH2O: 32.5μL</li>
+<br/>
+<li>Cycles: 25-35</li>
+<li>Pre-denaturation: 98℃,30s;</li>
+<li>Denaturation: 98℃,5-10s;</li>
+<li>Annealing: Depend on the primers, generally 45-65℃,10-30s;</li>
+<li>Extension: 72℃,20-30s/kb</li>
+<li>Fully extension: 72℃,2min</li>
+<li>Product Storage: 4℃</li>
+<li>Note: Different DNA polymerase has different protocol, this is only the case of Q5 DNA polymerase.</li>
+ </div>
+<a class="expand-btn7">Show More</a>
+		</div><!-- .entry-content -->
@@ Line 295: / Line 324: @@
-<!------------------------------------week6 end------------------------------------------------>
@@ Line 455: / Line 484: @@
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Difference between revisions of "Team:Tianjin/Protocol"

Revision as of 13:24, 4 October 2016

Plasmid Extraction

Agarose Gel Electrophoresis Products Recycling

Agarose Gel Electrophoresis

Restriction Endonuclease Digestion