Team:SYSU-China/Notebookt/Methods

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<DIV id="cont_column">
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<!--正 文 部 分 开 始-->
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<!--正文部分开始-->
<DIV class="chapter">
<DIV class="chapter">
-
<span>UPDATE <INS>09/18/2013</INS></span>
+
<span>Notebookt/Methods</span>
-
<h1> Molecular construction of the up stream elements(regulating parts,protein tTA,rtTA,tTA advanced ,and eGFP for contrast ) of tet systems. </h1>
+
 
-
<h2> Introduction</h2>
+
<h1>  
-
<p>
+
Methods
-
Team members in Charge:Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)
+
</h1>
-
</p>
+
<h2>  
-
<p>
+
Introduction
-
Author: Dawei He
+
-
</p>
+
-
<p>
+
-
Team members in Charge:Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)
+
-
</p>
+
-
<p>
+
-
Despite a good comprehension of the knowledge of Molecular biology and its relating experimental technologies,We never came to and stay in a Molecular biology lab and were completely lack of any experience. To achieve the designated tasks in extremely insufficient time(less than 3 months),We had to learn fast and start our work independently as soon as possible.
+
-
</p>
+
-
<h2> 2013.5.20-5.23
+
</h2>
</h2>
-
<p>
+
 
-
Contents: Contact and learning of Molecular construction
+
<p> Our experiments can be divided into two parts, one is the design and construction of genes and vectors, the other is test of parts and circuits with cells, including hepatoma cells, liver cells and iPSCs. So our protocol can be clearly be divided into these two parts, one for molecular operation, the other for cellular tests. Besides, quantitive measurement, like qPCR and western-blotting, is added in the molecular operation parts because it does not need cell operation.
</p>
</p>
-
<p>
+
<h2>  
-
Team members in Charge:Dawei He (2013.5.20-9.16)and Yiming Fang(2013 5.20-7.16)
+
Molecular Operation
-
</p>
+
-
<p>
+
-
1. DNA extraction techniques: Plasmid extraction, DNA clean up(extraction from PCR or restriction endonuclease digestion systems,extraction from Ag gel, etc) 
+
-
</p>
+
-
<p>
+
-
2. PCR techniques:
+
-
</p>
+
-
<p>
+
-
Primer(for molecular construction or sequencing) designing, PCR for molecular construction, colony PCR for verification.
+
-
</p>
+
-
<p>
+
-
3. Endonuclease digestion
+
-
</p>
+
-
<h2> 2013.5.24 to 5.31
+
</h2>
</h2>
-
<p>
+
 
-
Contents: First plasmid construction:pCDNA3.0-PGK-BSD
+
<p>< In the process of constructing circuit on vectors, PCR, PCR clean up, digestion of restriction endonuclease, gel extraction, ligation, transformation, colony PCR and plasmid extraction is implemented. Besides, for quantitive measurement, qPCR and western-blotting is used..</p>
 +
 
 +
<p><strong> PCR
 +
</strong> </p>
 +
 
 +
<p>
 +
1. Prepare the reaction mix:
</p>
</p>
-
<p>
+
<br><table width="400">
-
Annotation:pCDNA3.0 is a backbone plasmid ideal for transient expression in our designated cell lines including HEK293T,HepG2 and Bocs. All elements of our project are first to be incised into pCDNA3.0 and tested during transient expression in cell lines. Driven by promoter PGK,BSD express (!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!1)
+
<tr class="first-table-line">
-
</p>
+
<td>5x Fastpfu buffer </td>
-
<img src=" https://static.igem.org/mediawiki/2013/c/c0/Up_stream_elements_1.png " width="400" />
+
<td> 10ul </td>
 +
</tr>
 +
<tr>
 +
<td>2.5mM dNTPs  </td>
 +
<td>5ul </td>
 +
</tr>
 +
<tr>
 +
<td> Forward primer </td>
 +
<td>  0.8ul </td>
 +
</tr>
 +
<tr>
 +
<td> Reverse primer </td>
 +
<td>0.8ul </td>
 +
</tr>
 +
<tr>
 +
<td> Template DNA </td>
 +
<td> 40-50ng</td>
 +
</tr>
 +
<tr>
 +
<td>  FastPfu    </td>
 +
<td> 1ul </td>
 +
</tr>
 +
<tr>
 +
<td> ddH2O </td>
 +
<td> add to 50ul
 +
</td>
 +
</tr>
 +
</table><br />
<p>
<p>
-
Figure 1.Backbone plasmid pCDNA3.0(completed featured)
+
2. Set the program of thermal cycler
-
</p>
+
-
<img src=" https://static.igem.org/mediawiki/2013/a/a2/Up_stream_elements_2.png " width="400" />
+
-
<p>
+
-
Figure 2.pCDNA3.0-PGK-BSD
+
-
</p>
+
-
<p>
+
-
construction details:
+
</p>
</p>
 +
<br><table width="400">
-
<h3>1.PCR of PGK-BSD(915bp in length) from donor plasmid(as PCR template)
+
<tr class="first-table-line">
-
</h3>
+
<td>(1×) </td>
-
<p>
+
<td> 95℃  </td>
-
Primers:Forward primer with restriction site BamHI and necessary base pairs from 5’ end
+
<td>3min </td>
 +
</tr>
 +
<tr>
 +
<td>(25×)  </td>
 +
<td>95℃  </td>
 +
<td> 20s </td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td>  A-5℃ </td>
 +
<td> 20s (Tm≈A℃) </td>
 +
</tr>
 +
<tr>
 +
<td> </td>
 +
<td>  72℃</td>
 +
<td>30s for each 1kb </td>
 +
</tr>
 +
<tr>
 +
<td> (1×)</td>
 +
<td> 72℃ </td>
 +
<td>  5min</td>
 +
</tr>
 +
<tr>
 +
<td>(1×)  </td>
 +
<td> 4℃ </td>
 +
<td> forever </td>
 +
</tr>
 +
 
 +
 
 +
</table><br />
 +
<p>  
 +
3. Run the PCR program.
</p>
</p>
-
<p>
+
<p>  
-
        Reverse primer with restriction site XhoI and necessary base pairs from 5’ end
+
4. After PCR, load ~2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.
-
</p>
+
-
<p>
+
-
Bases pairs should appear at both flanks of restriction sites to provide anchoring sites for endonuclease,3’ end of the restriction sites in a primer is already covered by base pairs, so some base pairs must be added to its 5’ end. Without such manually added base pairs from 5’ end, efficiency of endonuclease digestion of PCR product will be significantly reduced.
+
</p>
</p>
-
<h3>
+
<p><strong> PCR clean up
-
1.1 PCR system set up
+
</strong></p>
-
</h3>
+
-
<table width="536">
+
-
<tr class="first-table-line">
+
-
<td>5×fastpfu PCR buffer </td>
+
-
<td>5μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>2.5mM dNTP:</td>
+
-
<td>2.5μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>10uM  PB  F primer </td>
+
-
<td>0.8μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>10uM  PB  R primer </td>
+
-
<td>0.8μl
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td> Template(plasmid)</td>
+
-
<td>4μl(5.36ng/ul,21.4ng in total. Acceptable quantity from 5-30ng)
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td> Fastpfu DNA polymerase </td>
+
-
<td>0.5μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td> ddH20</td>
+
-
<td>11.4μl(up to 25ul)</td>
+
-
</tr>
+
-
</table>
+
-
<h3>
+
-
1.2 temperature program set up
+
-
</h3>
+
-
<img src="https://static.igem.org/mediawiki/2013/d/dd/Up_stream_elements_3.png" width="400" />
 
<p>
<p>
-
Annealing temperature in main PCR cycles =Primer Tm-5℃
+
Use Anxygen PCR clean up kit for PCR clean up. In addition, restriction endonuclease digestion mix can be cleaned if the fragment to be deserted is less than 50bp.
</p>
</p>
<p>
<p>
-
Pre-cycles with lower annealing temperature in PCR process of primers with restriction sites(or any primers that include bases pairs not complementary to their original templates ) are necessary to achieve both specificity and quantity demand of PCR product.
+
1. Add 150ul of buffer PCR-A, vortex.  
</p>
</p>
-
 
-
 
<p>
<p>
-
During the first PCR cycle, primer binds only and incompletely to its original templates, so tm is lower compared to the one when primer binds total- complementarily to its product generated since the first cycle, which was why the idea of adding pre-cycles before main cycles came up. Although annealing temperature in pre-cycles can be calculated to details, the temperature 15℃ below annealing temperature in main cycles will be quite universal.
+
2. Pipette the liquid to a column, wait for 1min, then centrifuge 1000xg for 1min and 12000xg 30s. </p>
 +
<p>
 +
3. Use Buffer W2(700ul for the first step and 400ul again) to wash, 12000xg for 1min.  
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/4/42/Up_stream_elements_4.png" width="300" />
 
<p>
<p>
-
Primer binding to its original template
+
4. Dry the column on 65°C, warm the elutent in the meantime.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/5/53/Up_stream_elements_5.png" width="300" />
 
<p>
<p>
-
Primer binding to its PCR product
+
5. Use 25-30ul preheated elutent to wash DNA, wait for 1min and centrifuge 12000xg for 1min.
</p>
</p>
-
<h3>
 
-
1.3 Endonuclease digestion of backbone plasmid and insert fragment(PCR product)
 
-
</h3>
 
<p>
<p>
-
PCR product PGK-BSD was purified using PCR clean up kit.
+
6. Use Nanodrop to measure the concentration and quality of the DNA product.
</p>
</p>
-
<table width="536">
 
-
<tr class="first-table-line">
 
-
<td>10xFermentas Buffer G </td>
 
-
<td>3μL </td>
 
-
</tr>
 
-
<tr>
 
-
<td> pCDNA3.0</td>
 
-
<td>2μL(3500ng)</td>
 
-
</tr>
 
-
<tr>
 
-
<td> BamHI(Fer) (100% activity)</td>
 
-
<td>1μL </td>
 
-
</tr>
 
-
<tr>
 
-
<td> XhoI(Fer)(50-100% activity)</td>
 
-
<td> 2μL</td>
 
-
</tr>
 
-
<tr>
 
-
<td> ddH20 </td>
 
-
<td> 22μL(up to 30μL)</td>
 
-
</tr>
 
-
</table>
 
-
<table width="536">
 
-
<tr class="first-table-line">
 
-
<td>10xFermentas Buffer G </td>
 
-
<td>3μL </td>
 
-
</tr>
 
-
<tr>
 
-
<td> PGK-BSD </td>
 
-
<td>9μL(900ng)</td>
 
-
</tr>
 
-
<tr>
 
-
<td> BamHI(Fer) (100% activity)</td>
 
-
<td>1μL </td>
 
-
</tr>
 
-
<tr>
 
-
<td> XhoI(Fer)(50-100% activity)</td>
 
-
<td> 2μL</td>
 
-
</tr>
 
-
<tr>
 
-
<td> ddH20 </td>
 
-
<td> 15μL(up to 30μL)</td>
 
-
</tr>
 
-
</table>
 
-
<p>
 
-
Both digestion systems were incubated in 37℃ for 2h
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2013/archive/3/31/20130924110939%21Up_stream_elements_6.png" width="400" />
+
<p><strong> Digestion of restriction endonuclease
-
<p>
+
</strong></p>
-
Figure 3.</p>  
+
-
<p>
+
-
Result:backbone plasmid pCDNA3.0 was fully digested,but as the fragment excised from pCDNA3.0 was too short(which means too small in quality because it has the same amount of substance as the backbone’s) in length(less than 50bps),it cannot be identified in agarose gel-electrophoresis. When digested in a least one restriction site, plasmid turns from supercoil status to linearized and has lower mobility in gel-electrophoresis, so it can only be judged that backbone was definitely complete digested in at least one restriction site. Such assessment applies also to digestion of PCR product from whom the excised parts are almost never more than 50bps.
+
-
</p>  
+
-
 
-
<h3>
 
-
1.4 Ligation
 
-
</h3>
 
<p>
<p>
-
Ligation type:1 vector+1 insert fragment
+
NEB, Fermentas and Takara restriction enzymes are used. Before use, check the proper buffer for the enzyme, if the activity is low, add twice the amount but prevent the enzyme volume more than 10%, or star activity may rise.
</p>
</p>
<p>
<p>
-
Desired ratio of the amount of substance:
+
1. Set up the reaction mix:
-
</p>
+
-
<p>
+
-
            Vector:insert fragment =1:5
+
</p>
</p>
-
<table width="536">
+
<br><table width="400">
-
<tr class="first-table-line">
+
 
-
<td>10xFermentas T4 ligation buffer </td>
+
<tr class="first-table-line">
-
<td>2μL </td>
+
<td> DNA      </td>
-
</tr>
+
<td> 1ug for insert fragment, 3ug for vector </td>
 +
</tr>
<tr>
<tr>
-
<td> BamHI-(pCDNA3.0)-XhoI </td>
+
<td>10x buffer      </td>
-
<td>1μL(60ng)</td>
+
<td>2ul</td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td> BamHI-(PGK-BSD)-XhoI </td>
+
<td> Enzymes  </td>
-
<td>2μL(60ng)</td>
+
<td> 1ul each </td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td> Fermentas T4 ligase</td>
+
<td> ddH2O  </td>
-
<td> 1μL </td>
+
<td> add up to 20ul </td>
-
</tr>
+
</tr>
-
<tr>
+
</table><br />
-
<td> ddH20 </td>
+
-
<td>12μLup to 20μL)</td>
+
-
</tr>
+
-
</table>
+
<p>
<p>
-
Ligation for 2h at room temperature(25℃)
+
2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time 2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time .
</p>
</p>
-
 
-
<h3>
 
-
1.5 Transformation of ligation product
 
-
</h3>
 
-
 
<p>
<p>
-
Transformation system
+
3. Load 1.5ul mix on 1% agarose gel to have a quick run to check if the reaction is clear.
 +
</p>
 +
<p>
 +
4. 75°C water bath for 5min to stop the reaction.
</p>
</p>
-
<table width="536">
 
-
<tr class="first-table-line">
 
-
<td> DNA(ligation system)</td>
 
-
<td>2μL </td>
 
-
</tr>
 
-
<tr>
 
-
<td> Competent E.coli(Top10 strain)</td>
 
-
<td> 50μL </td>
 
-
</tr>
 
-
</table>
 
 +
<p><strong> Gel Extraction
 +
</strong></p>
<p>
<p>
-
After 2h ligation,ligation system,as well as Competent E.coli taken from -80℃ storage, was first incubated in ice for 5min before mixing together.  
+
Use Omega Gel Extraction Kit to do gel extraction.
</p>
</p>
<p>
<p>
-
Transformation system continued to be incubated in ice for 30mins,42℃heat shock for 60s,then again ice incubation for 5mins.
+
1. Cut the gel slice and weigh, 1ug for 1ul equal volume.  
</p>
</p>
<p>
<p>
-
Transformation system was added 600μL antibiotic-free LB medium and put in 37℃ thermostat shaker for 30mins-resuscitation(expression of Amp resistance protein)
+
2. Add 1x sample volume of Binding Buffer(XP2), add 1x sample volume of isopropanol if DNA is less than 500bp. Add 5ul of 5M pH5.2 NaAc when necessary.(When the color of liquid is orange or red, add it until it turns light yellow)
</p>
</p>
<p>
<p>
-
Transformation system was then centrifuged at 130,000g for 3mins.All but little supernatant was kept to suspend the E.coli,which was later transferred to the LB agar plate. The plate was put in 37℃ thermostat incubator overnight for colonies growth.
+
3. Incubate the mixture in 65°C for 7min or until totally dissolved. Vortex every 2-3min helps.
</p>
</p>
-
<h3>
 
-
1.6 Verification of the colonies(each as a single clone)
 
-
</h3>
 
-
<img src="https://2013.igem.org/File:Up_stream_elements_7.jpg" width="400" />
 
<p>
<p>
-
Figure 4.
+
4. Put it in room temperature to let it cool down to some extent(control the temperature before the liquid become solid). Load it into a column(for more than 700ul, load it in several times). Centrifuge 1000xg for 1min then 10000xg for 30s.  
</p>
</p>
<p>
<p>
-
colony PCR set up
+
5. Add 300ul Binding Buffer to wash the column, 10000xg 1min.
</p>
</p>
-
 
-
<table width="536">
 
-
<tr class="first-table-line">
 
-
<td>5×fastpfu PCR buffer </td>
 
-
<td>5μl </td>
 
-
</tr>
 
-
<tr>
 
-
<td>2.5mM dNTP</td>
 
-
<td>2.5μl </td>
 
-
</tr>
 
-
<tr>
 
-
<td>10uM  PGK-BSD  F primer  F primer </td>
 
-
<td>0.8μl </td>
 
-
</tr>
 
-
<tr>
 
-
<td>10uM  PGK-BSD  R primer </td>
 
-
<td>0.8μl
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td> Template (single-clone colony)</td>
 
-
<td>1μL(taken from the single-clone Colony suspended with 5μL ddH20
 
-
</td>
 
-
</tr>
 
-
<tr>
 
-
<td> Fastpfu DNA polymerase </td>
 
-
<td>0.5μl </td>
 
-
</tr>
 
-
<tr>
 
-
<td> ddH20</td>
 
-
<td>11.4μl(up to 25ul)</td>
 
-
</tr>
 
-
</table>
 
-
<img src="https://static.igem.org/mediawiki/2013/7/71/Up_stream_elements_8.png" width="400" />
 
<p>
<p>
-
Figure 5
+
6. Wash with buffer SPW, add 700ul, wait 2-3min and centrifuge 10000xg. Repeat 1 time.
</p>
</p>
<p>
<p>
-
Result:Clear band around 1000bp appeared for every 12 clone.
+
7. Discard the liquid and centrifuge 10000xg 2-3min to dry.
</p>
</p>
-
<h3>
 
-
1.7 Plasmid extraction and verification
 
-
</h3>
 
<p>
<p>
-
6 of the single-clone colonies were selected and added to 5ml Amp-LB medium and put to 37℃ thermostat shaker.
+
8. 65°C dry heat for elutent and column for totally dry. Wash it by 30-50ul elutent, centrifuge for 1min.
</p>
</p>
<p>
<p>
-
The bacteria were centrifuge after 16h-growth in the thermostat shaker.Plasmids were extracted using endotoxin-free kit.
+
9. Use Nanodrop to measure the concentration and quality of the DNA product.
</p>
</p>
 +
 +
<p>
<p>
-
The plasmids were later digested by BamHI and XhoI for verification.
+
Use Fermentas T4 ligase for ligation.
</p>
</p>
-
<table width="536">
+
<br><table width="400">
-
<tr class="first-table-line">
+
 
-
<td>10xFermentas Buffer G </td>
+
<tr class="first-table-line">
-
<td>2μL </td>
+
<td> Vector(about 5000bp)  </td>
-
</tr>
+
<td> 40ug </td>
 +
</tr>
<tr>
<tr>
-
<td> pCDNA3.0</td>
+
<td> Insert fragment </td>
-
<td>0.5μL(400ng)</td>
+
<td> 5x chemical amount of vector </td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td> BamHI(Fer) (100% activity)</td>
+
<td> T4 ligase buffer </td>
-
<td>0.3μL </td>
+
<td> 3ul </td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td> XhoI(Fer)(50-100% activity)</td>
+
<td> T4 ligase  </td>
-
<td>0.6μL</td>
+
<td>0.3-0.8ul </td>
-
</tr>
+
</tr>
-
<tr>
+
<tr>
-
<td> ddH20 </td>
+
<td> ddH2O </td>
-
<td> 16.6μL(up to 30μL)</td>
+
<td> add up to 30ul
-
</tr>
+
</td>
-
</table>
+
</tr>
-
<img src="https://static.igem.org/mediawiki/2013/5/57/Up_stream_elements_9.png" width="400" />
+
</table><br />
 +
 
<p>
<p>
-
Figure 6
+
For overnight ligation, use 0.3ul ligase, and for ligation that only proceeds in 2 hour, use 0.8ul ligase. The reaction temperature is room temperature, or a little bit lower than room temperature.(16°C)
</p>
</p>
 +
 +
<p><strong> Transformation</strong></p>
 +
<p>
<p>
-
Result: Clear band around 1000bp appeared. Plasmids from every 4 clones were verified.
+
1. Add 200ul TCM to whole reaction mix(for ligation mix or digestion mix), put on ice for 3-5min. This step is optional when plasmid is transformed.
</p>
</p>
<p>
<p>
-
Plasmids were later sent for sequencing
+
2. Add 50ul chemical competent cells(Top10)and mix gently. Incubate on ice for 30 min
</p>
</p>
-
<h2>
 
-
2013.6.1-6.14
 
-
</h2>
 
<p>
<p>
-
Construction of pCDNA3.0-pEF1a-tTA-PGK-BSD
+
3. Heat-shock in a 42℃ bath for 60 sec. Put it on ice for 3min.
</p>
</p>
<p>
<p>
-
            pCDNA3.0-pEF1a-rtTA-PGK-BSD
+
4. Add 0.5 ml LB/SOC(antibiotic free) medium and incubate at 37.0C for 30-60 min
</p>
</p>
<p>
<p>
-
pCDNA3.0-pEF1a-tTA advanced-PGK-BSD
+
5. Spin at 8,000*g for 3min. Decant most of LB medium but leave –100ul behind to resuspend the bug.
</p>
</p>
<p>
<p>
-
pCDNA3.0-pEF1a-eGFP-PGK-BSD
+
6. Plate all of the bug suspensions onto one LB(Amp+) plate. Mark the name of the plate and put it into 37°C incubator for 14-18h.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/7/75/Up_stream_elements_10.png" width="500" />
+
<p><strong>
-
<p>
+
Colony PCR
-
Figure.7
+
</strong></p>
-
</p>
+
-
<p>
+
-
PCR system set up
+
-
</p>
+
-
<table width="536">
+
-
<tr class="first-table-line">
+
-
<td>5×fastpfu PCR buffer </td>
+
-
<td>5μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>2.5mM dNTP:</td>
+
-
<td>2.5μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>10uM  PB  F primer </td>
+
-
<td>1.6μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td>10uM  PB  R primer </td>
+
-
<td>1.6μl
+
-
</td>
+
-
</tr>
+
-
<tr>
+
-
<td> Template(plasmid)</td>
+
-
<td>2ul(50ng)</td>
+
-
</tr>
+
-
<tr>
+
-
<td> Fastpfu DNA polymerase </td>
+
-
<td>1.0μl </td>
+
-
</tr>
+
-
<tr>
+
-
<td> ddH20</td>
+
-
<td>28.8μl(up to 25ul)</td>
+
-
</tr>
+
-
</table>
+
-
<p>
 
-
Temperature program set up
 
-
</p>
 
-
<table width="536">
 
-
<tr class="first-table-line">
 
-
<td>(1×) </td>
 
-
<td>95℃ </td>
 
-
<td>5min </td>
 
-
<td> initial denaturation </td>
 
-
</tr>
 
-
<tr>
 
-
<td>(5×)</td>
 
-
<td>95℃ </td>
 
-
<td>20s </td>
 
-
<td> denaturation </td>
 
-
</tr>
 
-
<tr>
 
-
<td>          </td>
 
-
<td>1.6μl  52℃ </td>
 
-
<td>20s </td>
 
-
<td> annealing </td>
 
-
</tr>
 
-
<tr>
 
-
<td> </td>
 
-
<td>  72℃ < /td>
 
-
        <td> 35s  </td>
 
-
      <td>elongation </td>
 
-
</tr>
 
-
<tr>
 
-
<td>(25×)
 
-
</td>
 
-
        <td>95℃ </td>
 
-
      <td> 20s  </td>
 
-
<td>denaturation </td>
 
-
</tr>
 
-
<tr>
 
-
<td>           
 
-
</td>
 
-
<td> 63℃ </td>
 
-
<td> 20s e </td>
 
-
<td>annealing </td>
 
-
</tr>
 
-
<tr>
 
-
<td>  </td>
 
-
<td>72℃ </td>
 
-
<td> 35s  </td>
 
-
<td> elongation </td>
 
-
</tr>
 
-
<tr>
 
-
<td>  72℃  </td>
 
-
<td>(1×)</td>
 
-
<td> 5min </td>
 
-
</tr>
 
-
<tr>
 
-
<td>(1×) 
 
-
</td>
 
-
<td> 4℃  </td>
 
-
<td> 10min </td>
 
-
</tr>
 
-
</table>
 
-
<img src="https://static.igem.org/mediawiki/2013/archive/d/da/20130924111810%21Up_stream_elements_11.png" width="400" />
 
<p>
<p>
-
Figure.8
+
Just like PCR protocol, but use takara rTaq enzyme.
</p>
</p>
 +
<p><strong>
 +
Plasmid Extraction
 +
</strong></p>
<p>
<p>
-
Digestions
+
Use Omega Endo-free kit for the plasmids for cell.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/4/49/Chart_1.png" width="300" />
 
-
<img src="https://static.igem.org/mediawiki/2013/f/fa/Chart_2.png" width="300" />
 
<p>
<p>
-
Incubated in 37℃ for 90mins
+
Use Anxygen Plasmid miniprep kit for plasmids for molecular cloning.
</p>
</p>
<p>
<p>
-
Ligation
+
Follow the protocals provided in the kit. But a 65°C elutent can cause higher efficiency.
</p>
</p>
 +
 +
 +
<h2>
 +
Cellular tests
 +
</h2>
 +
 +
 +
 +
<p>
<p>
-
Ligation type:1 vector+2 insert fragments
+
In the process of cellular tests, transient transfection, virus package, selecting stable cell line are included.
</p>
</p>
<p>
<p>
-
Desired ratio of the amount of substance:
+
<strong> Transient transfection
 +
</strong>
</p>
</p>
<p>
<p>
-
            Vector:insert fragment 1:insert fragment 2=1:3:3
+
1. Seed cells a night before.
</p>
</p>
<p>
<p>
-
Verification of single-clone colonies
+
2. Refresh medium right before transduction. Cell density should be around 60% at the time of transduction.
</p>
</p>
<p>
<p>
-
PCR verification of Result All chosen colonies are positive, so were the sequencing results.
+
3. For a single well in a 12-well plate, dilute 1.5ug plasmid DNA in 100ul opti-MEM and then add 4ul PEI.
</p>
</p>
-
<h2>
 
-
2013.6.18-7.1
 
-
</h2>
 
<p>
<p>
-
construction of pCDNA3.0-pCMV-RIP1
+
4. Gently mix and then incubate for 20min.
</p>
</p>
<p>
<p>
-
            pCDNA3.0-pCMV-RIP1-1X miR122 complete target
+
5. Add the mixture to cells and refresh after 6-8h.
</p>
</p>
<p>
<p>
-
pCDNA3.0-pCMV-RIP1-2X miR122 complete target
+
6. 1:1000 doxycycline is used to induce the Tet system.
</p>
</p>
-
<h2>
+
 
-
2013.7.5-7.31
+
-
</h2>
+
<p>
<p>
-
construction of  p199-pEF1a-tTA-PGK-BSD
+
<strong>
 +
Virus packaging
 +
</strong>
</p>
</p>
 +
 +
<p>
<p>
-
            p199-pEF1a-rtTA-PGK-BSD
+
1. seed cells a night before.
</p>
</p>
<p>
<p>
-
p199-pEF1a-tTA advanced-PGK-BSD
+
2. refresh medium right before transduction. Cell density should be 40% at the time of transduction.
</p>
</p>
<p>
<p>
-
p199-pEF1a-RIP1-2XmiR122 targets
+
3. For a single well in a 6-well plate, dilute totally 3ug plasmids in 100 ul CaCl2,gently mix.We use PCGP/VSVG to package retrovirus and PSPAX2/MD2G to package lentivirus.
</p>
</p>
 +
<p>
<p>
-
p199-pEF1a-RIP3-2XmiR122 targets
+
4. Add the mixture to 100ul BBS carefully and incubate for 20min.
</p>
</p>
<p>
<p>
-
p199-pEF1a-eGFP-PGK-BSD
+
5. Add the mixture to the cells and refresh after 6-8h.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/a/a7/Up_stream_elements_12.png" width="400" />
 
<p>
<p>
-
Figure.9
+
6. collect virus after 48h and store in -80°C.
</p>
</p>
-
<h2>
 
-
A major problem: DNA Recombination of pEF1a
 
-
2013.8.1-8.22
 
-
</h2>
 
<p>
<p>
-
Promoter EF1a we used to drive regulationg parts of tet-on or tet-off system are 537bp in length(supported by professor Lei xiao, Zhejiang University).By NCBI blast search,first 349bp of the 537bp-pEF1a was found to be CPPT,one of the essential elements of inserting backbone plasmid of lentivirus. While the function of CPPT cassette inside pEF1a is yet to figure out, the result of molecular construction suggested that CPPT, which exists both inside 537bp-pEF1a and p199 backbone plasmid, has high ratio of recombination that can interfere the normal inserting process of lentivirus vector and the expression of downstream genes.
+
7. For condensed virus, use 10cm culture medium and proportionally increase the reagents as described above. Medium containing virus is sealed by mineral oil and ultracentrifugate at 70000g for 1.5h. Collect the last 1ml medium and resuspend for further experiment.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/c/c6/Up_stream_elements_13.png" width="500" />
 
<p>
<p>
-
Figure 10. The recombination zone
+
<strong> Selecting stable cell line
 +
 
 +
</strong>
</p>
</p>
-
<p>
 
-
High-ratio recombination between the 2 copies CPPT in plasmid p199-pEF1a(537bp)-gene was first suspected as the forward sequencing primer often present 3 kinds of sequencing results:
 
-
1.No signal(most)    2.Double signal(few)      3.single signal(very few)
 
-
</p>
 
-
<img src=" https://static.igem.org/mediawiki/2013/8/8e/Up_stream_elements_14.png " width="500" />
 
-
<p>
 
-
Figure.11
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2013/a/ac/Chart_3.png" width="400" /> <p>
 
-
The recombination mechanism has it proof that the existence of type c plasmid was confirmed by reverse sequencing result (The sequence of some expressed genes driven by pEF1a are short enough to be covered and passed through by reverse sequencing),and that of type B plasmid was confirmed by double sequencing signal. Such recombination mechanism can also be supported by digestion of XhoI restriction site which exists right between CPPT and the 188-bp key region of pEF1a,and in the downstream of expressed genes.
 
-
</p>
 
-
<img src="https://static.igem.org/mediawiki/2013/c/cd/Up_stream_elements_15.png" width="400" /> </p>
 
-
                                             
 
-
<p>
 
-
 
-
<img src="https://2013.igem.org/File:Up_stream_elements_16.png" width="400" /> </p>
 
-
                                           
 
-
<p>
 
-
 
-
<img src="https://static.igem.org/mediawiki/2013/8/80/Up_stream_elements_17.png" width="300" />
 
<p>
<p>
-
Figure 11. (A).Hypothetical calculation of the result of digestion of XhoI restriction sites.(B).Agarose gel image of the result of digestion of XhoI restriction sites,and the verification of recombination
+
1. Seed cells a night before.
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/3/30/Chart_4.png" width="300" />
 
-
                       
 
<p>
<p>
-
According to the sequencing and digestion result, the recombination stopped in the downstream of CPPT cassette,unlike a dsDNA whose recombination will terminate at the end of the strain. Due to variable terminating position of recombination on our plasmid p199-537bp pEF1a, the impact on pEF1a and the expression of the genes driven can be different. However,as the first XhoI restriction site appears right between CPPT cassette and 170bps-key region( The 170bps region ,damage of which may has devastating impact on the expression of downstream genes,is found in almost every type of pEF1a), any recombination mode whose terminating point passes through the end of CPPT cassette will destroy XhoI site, therefore the XhoI digestion test(figure.11 B) is acute of any damaging recombination. The variable termination of recombination on our plasmid has its proof that some of plasmids with a single signal of forward sequencing, turned out to be recombined in XhoI digestion test.  
+
2. Refresh medium first.Add polybrene 8ul/ml final concentration. Gently add 100ul virus to every well in a 24-well plate. .
</p>
</p>
-
<img src="https://static.igem.org/mediawiki/2013/8/83/Up_stream_elements_18.png" width="400" />
 
<p>
<p>
-
Figure 12. Multiple recombination modes
+
3. Add 1:1000 blastisidin or 1:2000 2mg/ml puromycin to culture medium.
</p>
</p>
-
<h2>
 
-
Solutions
 
-
2013.8.1-8.22
 
-
</h2>
 
<p>
<p>
-
As the function of CPPT cassette inside pEF1a remains unclear,Several strategies were adopted to deal with the recombination of p199-537bp plasmid and keep such 537bps-pEF1a intact,but none of them has worked out ideally so far.
+
4. Three later, refresh culture medium and passage cells into new plates.
</p>
</p>
<p>
<p>
-
Strategy A
+
<strong> Basic cell culturing</strong>
</p>
</p>
<p>
<p>
-
Reconstruct all p199-537bp pEF1a and transport them into competent bacteria MDS42 strain whose genes expressing recombinases are completed knocked off, instead of Top10 strain.
+
Medium: 10% FBS+ 88% DMEM (high-glucose)+1% P/S (5000 mg/ml penicillin and 5000 mg/ml streptomycin)+1% L-glutamine (200 mM). Cells were passaged with 0.25%Trypsin.
</p>
</p>
<p>
<p>
-
Result: Failure in collecting single-clone colony, as the competence level of MDS42 strain is extreme low.
 
-
</p>
 
<p>
<p>
-
Stategy B
+
<p><strong>
-
</p>
+
hiPSC culturing
 +
</strong></p>
<p>
<p>
-
Pick up massive single-clone colonies(Top10) and conduct plasmids extraction
+
<strong>
 +
Introduction
 +
</strong>
</p>
</p>
<p>
<p>
-
    Result: All plasmid in XhoI digestion test is positive in recombination.
+
Culturing human and pluripotent stem cells requires the use of complex media and careful handling techniques. Here, we describe feeder-free and serum free way with the use of Gibco Essential 8 medium in combination with BD matrigel low growth factor to maintain and propagate high quality hIPSCs.</P>
</p>
</p>
 +
<p>The following protocol are modified from standard protocols from BD and Gibco </p>
<p>
<p>
-
Then we cutted the growth time in 37℃ thermostat shaker down to 8h in,as we hypothesized that the recombination process mainly occurred during bacteria growth in LB medium. But results were still the same.
+
 
 +
<strong>
 +
Materials required
 +
</strong>
</p>
</p>
<p>
<p>
-
Strategy C
+
Essential 8 medium, consisting of DMEM/F12(AM)1:1 and Essential 8 Supplement(50*)</p>
-
</p>
+
<p>
<p>
-
Pick up all colonies in the plate and concentrate them for direct plasmids extraction. The plasmids are then retransported  to MDS42 strain, whose single-clone colonies will be selected again for plasmids extraction.
+
0.5M EDTA, pH8.0</p><p>
 +
37°C water bath</p><p>
 +
BD matrigel low growh factor and Gibco knockout MEM</p><p>
 +
DPBS without Calcium and Magnesium</p><p>
 +
Approprate tissue culture plates and supplies</p><p>
 +
<strong> Preparing Media and Materials
 +
</strong>
</p>
</p>
<p>
<p>
-
Status: currently still underway.
+
<strong> Essential 8 Medium (500 mL of complete medium)
 +
</strong>
</p>
</p>
<p>
<p>
-
In such case , with the problem of recombination of 537bps-pEF1a unsolved, we chose to replace it with 188bps-pEF1a which was reported still possessing at least 30% of driving intensity of a full-length version pEF1a.And theoretically, the 188bps-pEF1a won’t be recombination capable.
+
1.  Thaw Essential 8 Supplement (50X) at 2–8°C overnight instead of at 37°C. </p><p>
-
</p>
+
2.  Mix the following component into 500 mL of complete Essential 8 Medium s: </p><p>
 +
DMEM/F-12 (HAM) 1:1            490 mL </p><p>
 +
Essential 8  Supplement (50X)      10 mL </p><p>
 +
Note: Before use, warm complete medium at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.  </p><p>
 +
<strong>0.5 mM EDTA in DPBS (50 mL)
 +
</strong>
 +
</p><p>
 +
1.  To prepare 50 mL of 0.5 mM EDTA in DPBS Mix the following components in a 50-mL conical tube in a biological safety cabinet to prepare 50 mL of 0.5 mM EDTA in DPBS: </p><p>
 +
DPBS without Calcium and Magnesium  50 mL </p><p>
 +
0.5 M EDTA                        50 µL </p><p>
 +
2.  Filter sterilize the solution. The solution can be stored at room temperature for up to six months. </p><p>
 +
<strong> Coating Culture Vessels with BD matrigel with low growth factor
 +
</strong>
 +
</p><p>
 +
BD Matrigel low growth factor Matrix should be aliquoted and frozen. Consult the Certificate of Analysis supplied with the BD Matrigel™ for the recommended aliquot size (“Dilution Factor”) to make up 25 mL of diluted matrix. Make sure to always keep BD Matrigel on ice when thawing and handling to prevent it from gelling. </p><p>
 +
Note: Use tissue culture-treated cultureware (e.g. 6-well plates, BD Catalog #353046). </p><p>
 +
1.  Thaw one aliquot of BD Matrigel on ice. </p><p>
 +
2.  Dispense 25 mL of cold dilution medium (DMEM/F-12; Catalog #36254) into a 50 mL conical tube and keep on ice. </p><p>
 +
3.  Add thawed BD Matrigel to the cold dilution medium (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired. </p><p>
 +
4.  Immediately use the diluted BD Matrigel solution to coat tissue culture-treated cultureware. for recommended coating volumes.  </p><p>
 +
<strong> Passaging iPSCs
 +
</strong>
 +
</p><p>
 +
In general, split cells when one of the following occurs: </p><p>
 +
•  PSC colonies are becoming too dense or too large.  </p><p>
 +
•  PSC colonies are showing increased differentiation. </p><p>
 +
•  The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days. </p><p>
 +
The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and </p><p>
 +
1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.  </p><p>
 +
•  A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.  </p><p>
 +
<strong> Passaging PSC Colonies using EDTA
 +
</strong>
 +
</p><p>
 +
Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages. </p><p>
 +
1.  Prior to starting, equilibrate your matrigel coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch. </p><p>
 +
Note: Do not warm medium in a 37°C water bath. </p><p>
 +
2.  Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes. </p><p>
 +
3.  Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Adjust the volume of EDTA for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface. </p><p>
 +
4.  Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel. </p><p>
 +
Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes. </p><p>
 +
5.  Aspirate the EDTA solution with a Pasteur pipette.  </p><p>
 +
6.  Add pre-warmed complete Essential 8 Medium to the dish. </p><p>
 +
7.  Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube. </p><p>
 +
For 6-well plate, 1ml/well 0.5nM EDTA in DPBS is used to digest cells and 2ml/well complete Essential 8 Medium is used for culturing. </p><p>
 +
Note: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping. </p><p>
 +
Note: Little or no extra pipetting is required to break up cell clumps after EDTA treatment. </p><p>
 +
Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the EDTA will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time. </p><p>
 +
8.  Aspirate residual matrigel solution from the pre-coated dish. </p><p>
 +
9.  Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels. </p><p>
 +
10.  Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels. </p><p>
 +
11.  Place dish gently into the 37°C, 5% CO 2 incubator and incubate the cells overnight. </p><p>
 +
12.  Feed PSC cells beginning the second day after splitting. Replace spent medium daily. </p><p>
 +
Note: It is normal to see cell debris and small colonies after passage.  </p><p>
 +
 
<p>
<p>
-
Currently we have successfully constructed p199-188bps pEF1a-tTA-PGK-BSD.
 
-
</p>
 
</DIV>
</DIV>
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</DIV>
</DIV>

Latest revision as of 01:29, 29 October 2013

ipsc

Notebookt/Methods

Methods

Introduction

Our experiments can be divided into two parts, one is the design and construction of genes and vectors, the other is test of parts and circuits with cells, including hepatoma cells, liver cells and iPSCs. So our protocol can be clearly be divided into these two parts, one for molecular operation, the other for cellular tests. Besides, quantitive measurement, like qPCR and western-blotting, is added in the molecular operation parts because it does not need cell operation.

Molecular Operation

< In the process of constructing circuit on vectors, PCR, PCR clean up, digestion of restriction endonuclease, gel extraction, ligation, transformation, colony PCR and plasmid extraction is implemented. Besides, for quantitive measurement, qPCR and western-blotting is used..

PCR

1. Prepare the reaction mix:


5x Fastpfu buffer 10ul
2.5mM dNTPs 5ul
Forward primer 0.8ul
Reverse primer 0.8ul
Template DNA 40-50ng
FastPfu 1ul
ddH2O add to 50ul

2. Set the program of thermal cycler


(1×) 95℃ 3min
(25×) 95℃ 20s
A-5℃ 20s (Tm≈A℃)
72℃ 30s for each 1kb
(1×) 72℃ 5min
(1×) 4℃ forever

3. Run the PCR program.

4. After PCR, load ~2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.

PCR clean up

Use Anxygen PCR clean up kit for PCR clean up. In addition, restriction endonuclease digestion mix can be cleaned if the fragment to be deserted is less than 50bp.

1. Add 150ul of buffer PCR-A, vortex.

2. Pipette the liquid to a column, wait for 1min, then centrifuge 1000xg for 1min and 12000xg 30s.

3. Use Buffer W2(700ul for the first step and 400ul again) to wash, 12000xg for 1min.

4. Dry the column on 65°C, warm the elutent in the meantime.

5. Use 25-30ul preheated elutent to wash DNA, wait for 1min and centrifuge 12000xg for 1min.

6. Use Nanodrop to measure the concentration and quality of the DNA product.

Digestion of restriction endonuclease

NEB, Fermentas and Takara restriction enzymes are used. Before use, check the proper buffer for the enzyme, if the activity is low, add twice the amount but prevent the enzyme volume more than 10%, or star activity may rise.

1. Set up the reaction mix:


DNA 1ug for insert fragment, 3ug for vector
10x buffer 2ul
Enzymes 1ul each
ddH2O add up to 20ul

2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time 2. Carefully mix the reaction mix and put it into 37°C water bath. Control the reaction time .

3. Load 1.5ul mix on 1% agarose gel to have a quick run to check if the reaction is clear.

4. 75°C water bath for 5min to stop the reaction.

Gel Extraction

Use Omega Gel Extraction Kit to do gel extraction.

1. Cut the gel slice and weigh, 1ug for 1ul equal volume.

2. Add 1x sample volume of Binding Buffer(XP2), add 1x sample volume of isopropanol if DNA is less than 500bp. Add 5ul of 5M pH5.2 NaAc when necessary.(When the color of liquid is orange or red, add it until it turns light yellow)

3. Incubate the mixture in 65°C for 7min or until totally dissolved. Vortex every 2-3min helps.

4. Put it in room temperature to let it cool down to some extent(control the temperature before the liquid become solid). Load it into a column(for more than 700ul, load it in several times). Centrifuge 1000xg for 1min then 10000xg for 30s.

5. Add 300ul Binding Buffer to wash the column, 10000xg 1min.

6. Wash with buffer SPW, add 700ul, wait 2-3min and centrifuge 10000xg. Repeat 1 time.

7. Discard the liquid and centrifuge 10000xg 2-3min to dry.

8. 65°C dry heat for elutent and column for totally dry. Wash it by 30-50ul elutent, centrifuge for 1min.

9. Use Nanodrop to measure the concentration and quality of the DNA product.

Use Fermentas T4 ligase for ligation.


Vector(about 5000bp) 40ug
Insert fragment 5x chemical amount of vector
T4 ligase buffer 3ul
T4 ligase 0.3-0.8ul
ddH2O add up to 30ul

For overnight ligation, use 0.3ul ligase, and for ligation that only proceeds in 2 hour, use 0.8ul ligase. The reaction temperature is room temperature, or a little bit lower than room temperature.(16°C)

Transformation

1. Add 200ul TCM to whole reaction mix(for ligation mix or digestion mix), put on ice for 3-5min. This step is optional when plasmid is transformed.

2. Add 50ul chemical competent cells(Top10)and mix gently. Incubate on ice for 30 min

3. Heat-shock in a 42℃ bath for 60 sec. Put it on ice for 3min.

4. Add 0.5 ml LB/SOC(antibiotic free) medium and incubate at 37.0C for 30-60 min

5. Spin at 8,000*g for 3min. Decant most of LB medium but leave –100ul behind to resuspend the bug.

6. Plate all of the bug suspensions onto one LB(Amp+) plate. Mark the name of the plate and put it into 37°C incubator for 14-18h.

Colony PCR

Just like PCR protocol, but use takara rTaq enzyme.

Plasmid Extraction

Use Omega Endo-free kit for the plasmids for cell.

Use Anxygen Plasmid miniprep kit for plasmids for molecular cloning.

Follow the protocals provided in the kit. But a 65°C elutent can cause higher efficiency.

Cellular tests

In the process of cellular tests, transient transfection, virus package, selecting stable cell line are included.

Transient transfection

1. Seed cells a night before.

2. Refresh medium right before transduction. Cell density should be around 60% at the time of transduction.

3. For a single well in a 12-well plate, dilute 1.5ug plasmid DNA in 100ul opti-MEM and then add 4ul PEI.

4. Gently mix and then incubate for 20min.

5. Add the mixture to cells and refresh after 6-8h.

6. 1:1000 doxycycline is used to induce the Tet system.

Virus packaging

1. seed cells a night before.

2. refresh medium right before transduction. Cell density should be 40% at the time of transduction.

3. For a single well in a 6-well plate, dilute totally 3ug plasmids in 100 ul CaCl2,gently mix.We use PCGP/VSVG to package retrovirus and PSPAX2/MD2G to package lentivirus.

4. Add the mixture to 100ul BBS carefully and incubate for 20min.

5. Add the mixture to the cells and refresh after 6-8h.

6. collect virus after 48h and store in -80°C.

7. For condensed virus, use 10cm culture medium and proportionally increase the reagents as described above. Medium containing virus is sealed by mineral oil and ultracentrifugate at 70000g for 1.5h. Collect the last 1ml medium and resuspend for further experiment.

Selecting stable cell line

1. Seed cells a night before.

2. Refresh medium first.Add polybrene 8ul/ml final concentration. Gently add 100ul virus to every well in a 24-well plate. .

3. Add 1:1000 blastisidin or 1:2000 2mg/ml puromycin to culture medium.

4. Three later, refresh culture medium and passage cells into new plates.

Basic cell culturing

Medium: 10% FBS+ 88% DMEM (high-glucose)+1% P/S (5000 mg/ml penicillin and 5000 mg/ml streptomycin)+1% L-glutamine (200 mM). Cells were passaged with 0.25%Trypsin.

hiPSC culturing

Introduction

Culturing human and pluripotent stem cells requires the use of complex media and careful handling techniques. Here, we describe feeder-free and serum free way with the use of Gibco Essential 8 medium in combination with BD matrigel low growth factor to maintain and propagate high quality hIPSCs.

The following protocol are modified from standard protocols from BD and Gibco

Materials required

Essential 8 medium, consisting of DMEM/F12(AM)1:1 and Essential 8 Supplement(50*)

0.5M EDTA, pH8.0

37°C water bath

BD matrigel low growh factor and Gibco knockout MEM

DPBS without Calcium and Magnesium

Approprate tissue culture plates and supplies

Preparing Media and Materials

Essential 8 Medium (500 mL of complete medium)

1. Thaw Essential 8 Supplement (50X) at 2–8°C overnight instead of at 37°C.

2. Mix the following component into 500 mL of complete Essential 8 Medium s:

DMEM/F-12 (HAM) 1:1 490 mL

Essential 8 Supplement (50X) 10 mL

Note: Before use, warm complete medium at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

0.5 mM EDTA in DPBS (50 mL)

1. To prepare 50 mL of 0.5 mM EDTA in DPBS Mix the following components in a 50-mL conical tube in a biological safety cabinet to prepare 50 mL of 0.5 mM EDTA in DPBS:

DPBS without Calcium and Magnesium 50 mL

0.5 M EDTA 50 µL

2. Filter sterilize the solution. The solution can be stored at room temperature for up to six months.

Coating Culture Vessels with BD matrigel with low growth factor

BD Matrigel low growth factor Matrix should be aliquoted and frozen. Consult the Certificate of Analysis supplied with the BD Matrigel™ for the recommended aliquot size (“Dilution Factor”) to make up 25 mL of diluted matrix. Make sure to always keep BD Matrigel on ice when thawing and handling to prevent it from gelling.

Note: Use tissue culture-treated cultureware (e.g. 6-well plates, BD Catalog #353046).

1. Thaw one aliquot of BD Matrigel on ice.

2. Dispense 25 mL of cold dilution medium (DMEM/F-12; Catalog #36254) into a 50 mL conical tube and keep on ice.

3. Add thawed BD Matrigel to the cold dilution medium (in the 50 mL tube) and mix well. The vial may be washed with cold medium if desired.

4. Immediately use the diluted BD Matrigel solution to coat tissue culture-treated cultureware. for recommended coating volumes.

Passaging iPSCs

In general, split cells when one of the following occurs:

• PSC colonies are becoming too dense or too large.

• PSC colonies are showing increased differentiation.

• The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days.

The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and

1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.

• A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.

Passaging PSC Colonies using EDTA

Note: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells the overall culture health should improve throughout the early passages.

1. Prior to starting, equilibrate your matrigel coated dishes to room temperature in the hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch.

Note: Do not warm medium in a 37°C water bath.

2. Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with Dulbecco’s PBS (DPBS) without Calcium and Magnesium. Refer to Table 2 for the recommended volumes.

3. Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Adjust the volume of EDTA for various dish sizes (refer to Table 2). Swirl the dish to coat the entire cell surface.

4. Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies will appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel.

Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.

5. Aspirate the EDTA solution with a Pasteur pipette.

6. Add pre-warmed complete Essential 8 Medium to the dish.

7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL glass pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube.

For 6-well plate, 1ml/well 0.5nM EDTA in DPBS is used to digest cells and 2ml/well complete Essential 8 Medium is used for culturing.

Note: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping.

Note: Little or no extra pipetting is required to break up cell clumps after EDTA treatment.

Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the EDTA will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time.

8. Aspirate residual matrigel solution from the pre-coated dish.

9. Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2 for volumes for other culture vessels.

10. Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.

11. Place dish gently into the 37°C, 5% CO 2 incubator and incubate the cells overnight.

12. Feed PSC cells beginning the second day after splitting. Replace spent medium daily.

Note: It is normal to see cell debris and small colonies after passage.

Sun Yat-Sen University, Guangzhou, China

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