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<p>30 cycles(step 2 ~ step 4)</p> | <p>30 cycles(step 2 ~ step 4)</p> | ||
<p>3.Agarose gel electrophoresis Result:</p> | <p>3.Agarose gel electrophoresis Result:</p> | ||
− | <p><img src="https:// | + | <p><img src="https://static.igem.org/mediawiki/2016/4/49/T--USTC--NB_0729_0804_16.jpeg" /> |
(For the two pictures,the left four are AD,the right four are BD.)</p> | (For the two pictures,the left four are AD,the right four are BD.)</p> | ||
<p>4.Then we did gel extration.The result is as follows:</p> | <p>4.Then we did gel extration.The result is as follows:</p> | ||
Line 391: | Line 391: | ||
1.5 μL PstI.</p> | 1.5 μL PstI.</p> | ||
<p>Agarose gel electrophoresis Result:</p> | <p>Agarose gel electrophoresis Result:</p> | ||
− | <p><img src="https:// | + | <p><img src="https://static.igem.org/mediawiki/2016/8/83/T--USTC--NB_0729_0804_17.jpeg" alt="图片名称" /></p> |
<p>We did gel extration.The result are as follows:</p> | <p>We did gel extration.The result are as follows:</p> | ||
<table class="ui celled table"> | <table class="ui celled table"> | ||
Line 425: | Line 425: | ||
Use clean water,clean TAE buffer,clean erlenmeyer flask to make superclean gel.</p> | Use clean water,clean TAE buffer,clean erlenmeyer flask to make superclean gel.</p> | ||
<p>Agarose gel electrophoresis Result:</p> | <p>Agarose gel electrophoresis Result:</p> | ||
− | <p><img src="https:// | + | <p><img src="https://static.igem.org/mediawiki/2016/1/1d/T--USTC--NB_0729_0804_18.jpeg" alt="图片名称" /></p> |
<p>We did gel extration. | <p>We did gel extration. | ||
<strong>Special notes:</strong> | <strong>Special notes:</strong> | ||
Line 569: | Line 569: | ||
1 μL PstI.</p> | 1 μL PstI.</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--USTC--NB_0729_0804_19.jpeg" alt="图片名称" /></p> |
<p>We did gel extration.The result are as follows:</p> | <p>We did gel extration.The result are as follows:</p> | ||
<p>first elution:</p> | <p>first elution:</p> | ||
Line 698: | Line 698: | ||
<p>35 cycles(step 2 ~ step 4)</p> | <p>35 cycles(step 2 ~ step 4)</p> | ||
<p>4.Agarose gel electrophoresis Result: | <p>4.Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/c/c9/T--USTC--NB_0729_0804_20.jpeg" alt="图片名称" /></p> |
<p>5.Conclusion: | <p>5.Conclusion: | ||
In the result of agarose gel electophoresis, we can see that the band is at a lower place than where it should be. But we still assume this is the band of SUP35, and keep doing this same experiment to check whether this is the SUP35 or not.</p> | In the result of agarose gel electophoresis, we can see that the band is at a lower place than where it should be. But we still assume this is the band of SUP35, and keep doing this same experiment to check whether this is the SUP35 or not.</p> | ||
Line 741: | Line 741: | ||
35 cycles(step 2 ~ step 4)</p> | 35 cycles(step 2 ~ step 4)</p> | ||
<p>4.Agarose gel electrophoresis Result: | <p>4.Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/thumb/d/d0/T--USTC--NB_0729_0804_22.jpeg/750px-T--USTC--NB_0729_0804_22.jpeg" alt="图片名称" /></p> |
<p><strong>Plasmid Extraction for sfGFP1-10</strong> | <p><strong>Plasmid Extraction for sfGFP1-10</strong> | ||
**Recorder: Yin Wu,Wenkai Han **</p> | **Recorder: Yin Wu,Wenkai Han **</p> | ||
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<p><strong>Agarose gel electrophoresis gel</strong> | <p><strong>Agarose gel electrophoresis gel</strong> | ||
Agarose gel electrophoresis Result: | Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/a/a6/T--USTC--NB_0729_0804_23.jpeg" alt="图片名称" /></p> |
<p><strong>Recorder: Ya Jiang, Yin Wu</strong> | <p><strong>Recorder: Ya Jiang, Yin Wu</strong> | ||
<strong>Transformation of recombinant plasmid of sfGFP1-10 and PYescGAP</strong></p> | <strong>Transformation of recombinant plasmid of sfGFP1-10 and PYescGAP</strong></p> | ||
Line 846: | Line 846: | ||
35 cycles(step 2 ~ step 4)</p> | 35 cycles(step 2 ~ step 4)</p> | ||
<p>4.Agarose gel electrophoresis Result: | <p>4.Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/0/08/T--USTC--NB_0729_0804_24.jpeg" alt="图片名称" /></p> |
<p>5.Gel Extraction of PCR product of SUP35: | <p>5.Gel Extraction of PCR product of SUP35: | ||
|sample|1|2|3|4| | |sample|1|2|3|4| | ||
Line 862: | Line 862: | ||
1 μL PstI.</p> | 1 μL PstI.</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/e/ed/T--USTC--NB_0729_0804_25.jpeg" alt="图片名称" /></p> |
<p>It shows the DNA travels very slow in the gel because of its high dencity.It tells us not to use the gel which has been put for a really long time.The experiment failed,so we didn't do gel extraction.</p> | <p>It shows the DNA travels very slow in the gel because of its high dencity.It tells us not to use the gel which has been put for a really long time.The experiment failed,so we didn't do gel extraction.</p> | ||
<p><strong>Recorder:Chengle Zhang</strong> | <p><strong>Recorder:Chengle Zhang</strong> | ||
Line 937: | Line 937: | ||
Mix gently and incubate at 37 degree Celsius for 15 min. | Mix gently and incubate at 37 degree Celsius for 15 min. | ||
Agarose gel electrophoresis Results: | Agarose gel electrophoresis Results: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/b/b3/T--USTC--NB_0729_0804_27.jpeg" alt="图片名称" /></p> |
<table class="ui celled table"> | <table class="ui celled table"> | ||
<thead> | <thead> | ||
Line 1,046: | Line 1,046: | ||
<p>Mix gently and incubate at 37 degree Celsius for 30 mins .</p> | <p>Mix gently and incubate at 37 degree Celsius for 30 mins .</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/7/7c/T--USTC--NB_0729_0804_28.jpeg" alt="图片名称" /> |
(from left to right:marker(Transplus 2K),sample1,,sample2,sample3)</p> | (from left to right:marker(Transplus 2K),sample1,,sample2,sample3)</p> | ||
<p>We did gel extration.The result are as follows:</p> | <p>We did gel extration.The result are as follows:</p> | ||
Line 1,087: | Line 1,087: | ||
0.4 μL PstI.</p> | 0.4 μL PstI.</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/e/ec/T--USTC--NB_0729_0804_29.jpeg" alt="图片名称" /> |
(from left to right:marker(Transplus 2K),sample1,sample1,control1,sample2,sample2,control2)</p> | (from left to right:marker(Transplus 2K),sample1,sample1,control1,sample2,sample2,control2)</p> | ||
<p>We did gel extration.The result are as follows:</p> | <p>We did gel extration.The result are as follows:</p> | ||
Line 1,115: | Line 1,115: | ||
<strong>Colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD</strong></p> | <strong>Colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD</strong></p> | ||
<p>We did colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.Then we did PCR with primer designed by ourselves(biobrick-p/r).The agarose gel electrophoresis result is as follows: | <p>We did colony picking of combinant plasmid of PYescGAP and AD,PYescGAP and BD.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.Then we did PCR with primer designed by ourselves(biobrick-p/r).The agarose gel electrophoresis result is as follows: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/5/53/T--USTC--NB_0729_0804_30.jpeg" alt="图片名称" /> |
(from left to right:AD-1,AD-2,AD-3,AD-control,BD-1,BD-2,BD-3,BD-control) | (from left to right:AD-1,AD-2,AD-3,AD-control,BD-1,BD-2,BD-3,BD-control) | ||
It shows that the colonies we picked are not successful ones.</p> | It shows that the colonies we picked are not successful ones.</p> | ||
Line 1,192: | Line 1,192: | ||
<p>Mix gently and incubate at 37 degree Celsius for 30 mins .</p> | <p>Mix gently and incubate at 37 degree Celsius for 30 mins .</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/4/46/T--USTC--NB_0729_0804_31.jpeg" alt="图片名称" /> |
(from left to right:marker(Transplus 2K),sample1,,sample2,sample3,control-1,sample4,sample5,sample6,control-2)</p> | (from left to right:marker(Transplus 2K),sample1,,sample2,sample3,control-1,sample4,sample5,sample6,control-2)</p> | ||
<p>We did gel extration.The result are as follows:</p> | <p>We did gel extration.The result are as follows:</p> | ||
Line 1,353: | Line 1,353: | ||
<p>30 cycles(step 2 ~ step 4)</p> | <p>30 cycles(step 2 ~ step 4)</p> | ||
<p>The agarose gel electrophoresis result is as follows: | <p>The agarose gel electrophoresis result is as follows: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/4/4d/T--USTC--NB_0729_0804_32.jpeg" alt="图片名称" /></p> |
<p><strong>Recorder: Yu Xie</strong> | <p><strong>Recorder: Yu Xie</strong> | ||
<strong>Ligation of PYescGAP and PRsfGFP1-10</strong></p> | <strong>Ligation of PYescGAP and PRsfGFP1-10</strong></p> | ||
Line 1,448: | Line 1,448: | ||
0 μL Sterilized ddH<sub>2</sub>O.</p> | 0 μL Sterilized ddH<sub>2</sub>O.</p> | ||
<p>Agarose gel electrophoresis Result: | <p>Agarose gel electrophoresis Result: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/8/88/T--USTC--NB_0729_0804_33.jpeg" alt="图片名称" /> |
(PS: 1 to 4 are the digest products, 5 is the control group) | (PS: 1 to 4 are the digest products, 5 is the control group) | ||
We did gel extration.</p> | We did gel extration.</p> | ||
Line 1,481: | Line 1,481: | ||
<strong>Check again for the last picking colony of combinant plasmid of PYescGAP and AD,PYescGAP and BD</strong></p> | <strong>Check again for the last picking colony of combinant plasmid of PYescGAP and AD,PYescGAP and BD</strong></p> | ||
<p>The agarose gel electrophoresis result is as follows: | <p>The agarose gel electrophoresis result is as follows: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/5/5e/T--USTC--NB_0729_0804_34.jpeg" alt="图片名称" /> |
(from left to right:AD-cut,AD-PCR,AD-1-1,AD-1-2,AD-1-3,AD-2-1,AD-2-2,AD-2-3,BD-cut,BD-PCR,BD-1-1,BD-1-2,BD-1-3,BD-2-1,BD-2-2,BD-2-3) | (from left to right:AD-cut,AD-PCR,AD-1-1,AD-1-2,AD-1-3,AD-2-1,AD-2-2,AD-2-3,BD-cut,BD-PCR,BD-1-1,BD-1-2,BD-1-3,BD-2-1,BD-2-2,BD-2-3) | ||
It still shows that the colonies we picked are not successful ones.</p> | It still shows that the colonies we picked are not successful ones.</p> |
Revision as of 17:15, 19 October 2016
Notebook
Together we stand
Performers
![](https://static.igem.org/mediawiki/2016/b/b3/T--USTC--TeamPhoto.jpeg)
Date: 8.5
Plasmid Extraction for pGal1-GFP Recorder: Yin Wu and Kaiyue Ma
sample:bacteria containing pGal1-GFP
number | 1-1 | 1-2 | 1-3 | 2-1 | 2-2 | 2-3 |
---|---|---|---|---|---|---|
A260/A280 | 1.88 | 1.89 | 1.89 | 1.81 | 1.90 | 1.89 |
concentration(ng/μL) | 196.0 | 191.9 | 116.5 | 250.4 | 189.5 | 157.5 |
stage | temperature | time | ||||
---|---|---|---|---|---|---|
step 1 | 95 | 10 min | ||||
step 2 | 95 | 1 min | ||||
step 3 | 57 | 1 min | ||||
step 4 | 72 | 54 s | ||||
step 5 | 72 | 10 min | ||||
step 6 | 4 | -- |
sample | AD4 | AD5 | BD2 | BD4 | ||
---|---|---|---|---|---|---|
A260/A280 | 1.88 | 1.89 | 1.90 | 1.89 | ||
concentration(ng/μL) | 196.1 | 84.6 | 111.1 | 152.1 |
sample | pGal1-GFP1 | pGal1-GFP2 | ||||
---|---|---|---|---|---|---|
A260/A280 | 1.97 | 1.98 | ||||
concentration(ng/μL) | 7.6 | 8.8 |
sample | AD-1 | |||||
---|---|---|---|---|---|---|
A260/A280 | 1.90 | |||||
A260/A230 | 0.29 | |||||
concentration(ng/μL) | 118.6 |
sample | AD-2 | |||||
---|---|---|---|---|---|---|
A260/A280 | 1.82 | |||||
A260/A230 | 0.82 | |||||
concentration(ng/μL) | 70.5 |
sample | AD-3 | |||||
---|---|---|---|---|---|---|
A260/A280 | 1.90 | |||||
A260/A230 | 0.44 | |||||
concentration(ng/μL) | 22.0 |
sample | AD-4 | |||||
---|---|---|---|---|---|---|
A260/A280 | 1.76 | |||||
A260/A230 | 0.46 | |||||
concentration(ng/μL) | 19.9 |
sample | 1 | 2 | 3 | 4 | ||
---|---|---|---|---|---|---|
A260/A230 | 0.02 | 0.09 | 0.01 | 0.19 | ||
A260/A280 | 1.93 | 1.93 | 1.95 | 1.92 | ||
concentration(ng/μL) | 95.8 | 125.0 | 14.5 | 17.4 |
sample | BD | pGal1&GFP | ||||
---|---|---|---|---|---|---|
A260/A280 | 1.90 | 1.94 | ||||
A260/A230 | 0.62 | 0.94 | ||||
concentration(ng/μL) | 92.9 | 29.1 |
sample | BD | pGal1&GFP | ||||
---|---|---|---|---|---|---|
A260/A280 | 1.99 | 1.94 | ||||
A260/A230 | 0.27 | 1.04 | ||||
concentration(ng/μL) | 24.5 | 12.0 |
primer | 1 | 2 | ||||
---|---|---|---|---|---|---|
sequence | 5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGATT CAAACCAA GGCAACA-3' |
5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTTAA CAACTTCG TCATCCACTTCT-3' |
stage | temperature | time | ||||
---|---|---|---|---|---|---|
step 1 | 95 | 7 min | ||||
step 2 | 95 | 10 s | ||||
step 3 | 57 | 15 s | ||||
step 4 | 72 | 50 s | ||||
step 5 | 72 | 10 min | ||||
step 6 | 4 | -- |
primer | 1 | 2 | ||||
---|---|---|---|---|---|---|
sequence | 5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGA TTCAAACCA AGGCAACA-3' |
5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTT AACAACTTC GTCATCCACT TCT-3' |
number | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|
A260/A280 | 1.87 | 1.88 | 1.89 | 1.87 | 1.87 | 1.86 |
concentration(ng/μL) | 344.5 | 282.7 | 244.5 | 327.3 | 380.5 | 473.6 |
sample | 1 | 2 | ||||
---|---|---|---|---|---|---|
A260/A280 | 1.87 | 1.90 | ||||
concentration(ng/μL) | 90.4 | 100.5 |
primer | 1 | 2 | ||||
---|---|---|---|---|---|---|
sequence | 5'-CCGGAAT TCGCGGCC GCTTCTAGA TGGGCGA TTCAAACCA AGGCAACA-3' |
5'-TGCACTG CAGCGGCC GCTACTAGT AATCGTT AACAACTTC GTCATCCACT TCT-3' |
sample | sfGFP11-5 | sfGFP11-6 | sfGFP11-7 | sfGFP11-8 | ||
---|---|---|---|---|---|---|
A260/A280 | 1.84 | 1.93 | 1.71 | 1.55 | ||
A260/A230 | 1.79 | 2.51 | 1.04 | 0.56 | ||
concentration(ng/μL) | 49.2 | 68.7 | 14.5 | 36.3 |
sample | sfGFP11 | pSB1A3 | ||||
---|---|---|---|---|---|---|
A260/A230 | 0.07 | 0.08 | ||||
A260/A280 | 2.03 | 1.83 | ||||
concentration(ng/μL) | 14.0 | 12.5 |
sample | 3-2 | 4-1 | 4-2 | 5-1 | 5-2 | 3-1 |
---|---|---|---|---|---|---|
A260/A280 | 1.71 | 1.77 | 1.87 | 1.85 | 1.89 | 1.86 |
A260/A230 | 0.89 | 1.26 | 2.37 | 2.23 | 2.27 | 2.14 |
concentration(ng/μL) | 49.2 | 251.1 | 84.2 | 649.3 | 111.4 | 260.9 |
sample | 1 | 2 | ||||
---|---|---|---|---|---|---|
A260/A280 | 1.83 | 1.90 | ||||
A260/A230 | 1.94 | 2.03 | ||||
concentration(ng/μL) | 68.2 | 49.3 |
sample | 1 | 2 | 3 | |||
---|---|---|---|---|---|---|
A260/A280 | 1.86 | 1.82 | 1.72 | |||
A260/A230 | 0.81 | 0.42 | 0.06 | |||
concentration(ng/μL) | 98.4 | 73.7 | 16.5 |
group | 1 | |||||
---|---|---|---|---|---|---|
A260/A280 | 1.89 | |||||
A260/A230 | 0.41 | |||||
concentration(ng/μL) | 30.2 |
Sample | 24μL sfGFP11-1 | 24μL sfGFP11-2 | 20μL sfGFP11-3 | 18μL pSB1C3-PRsfGFP1-10-1 | 18μL pSB1C3-PRsfGFP1-10-2 | 8μL pSB1C3-PRsfGFP1-10-3 |
---|---|---|---|---|---|---|
nuclease-free water(μL) | 0 | 0 | 4 | 6 | 6 | 8 |
fastdigest buffer(μL) | 3 | 3 | 3 | 3 | 3 | 2 |
PstI(μL) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1 |
XbaI(μL) | 1.5 | 1.5 | 1.5 | 0 | 0 | 0 |
EcoRI(μL) | 0 | 0 | 0 | 1.5 | 1.5 | 1 |
total(μL) | 30 | 30 | 30 | 30 | 30 | 20 |
sample | pSB1C3-PRsfGFP1-10-1 | pSB1C3-PRsfGFP1-10-2 | |||
---|---|---|---|---|---|
A260/A230 | 0.56 | 0.33 | |||
A260/A280 | 1.87 | 1.79 | |||
concentration(ng/μL) | 202.7 | 60.7 |
sample | pSB1C3-1 | pSB1C3-2 | YeTGAP | YeTGAP | YeTGAP |
---|---|---|---|---|---|
A260/A280 | 1.91 | 1.85 | 1.89 | 1.86 | 1.88 |
concentration(ng/μL) | 300.0 | 580.4 | 993.5 | 593.0 | 968.1 |
sample | 1-4 | |||
---|---|---|---|---|
Sterilized ddH2O | 32.5 μL | |||
5×PrimeSTAR Buffer (Mg2+Plus) | 10 μL | |||
dNTP Mixture (2.5 mM each) | 4 μL | |||
Template (sfGFP) | 1 μL | |||
standard-biobrick-p | 1 μL | |||
standard-biobrick-r | 1 μL | |||
PrimeSTAR HS DNA Polymerase (2.5 U/μl) | 0.5 μL | |||
total | 50 μL |
stage | temperature | time | ||
---|---|---|---|---|
step 1 | 95 | 5 min | ||
step 2 | 95 | 10 s | ||
step 3 | 55 | 15 s | ||
step 4 | 72 | 12 s | ||
step 5 | 72 | 10 min | ||
step 6 | 4 | -- |
Sample | volume | |||
---|---|---|---|---|
PYescGAP (cut) | 1.5μL | |||
PRsfGFP1-10 | 0.5μL | |||
nuclease free water | 15.7μL | |||
10* T4 DNA Ligase Buffer | 2.0μL | |||
T4 DNA Ligase | 0.3μL | |||
total | 20μL |
sample | 1 | 2 | 3 | 4 |
---|---|---|---|---|
A260/A280 | 1.91 | 1.94 | 1.91 | 1.89 |
A260/A230 | 1.99 | 2.06 | 1.22 | 1.72 |
concentration(ng/μL) | 84.6 | 74.7 | 86.9 | 104.4 |
sample | 1 |
---|---|
A260/A280 | 2.12 |
A260/A230 | 0.04 |
concentration(ng/μL) | 34.6 |