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<p>30 cycles(Duration ~ Extend)</p> | <p>30 cycles(Duration ~ Extend)</p> | ||
<p>Agarose gel electrophoresis Results: | <p>Agarose gel electrophoresis Results: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/3/30/T--USTC--NB_0811_5.jpeg" alt="图片名称" /> |
Agarose gel electrophoresis of PCR product | Agarose gel electrophoresis of PCR product | ||
(from left to right:sample 4,3,2,1,marker)</p> | (from left to right:sample 4,3,2,1,marker)</p> | ||
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<p>30 cycles(Duration ~ Extend)</p> | <p>30 cycles(Duration ~ Extend)</p> | ||
<p>Agarose gel electrophoresis Results: | <p>Agarose gel electrophoresis Results: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/4/4a/T--USTC--NB_0811_6.jpeg" alt="图片名称" /> |
Agarose gel electrophoresis of PCR product | Agarose gel electrophoresis of PCR product | ||
(from left to right:marker, sample 1,2,3,4)</p> | (from left to right:marker, sample 1,2,3,4)</p> | ||
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<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/f/ff/T--USTC--NB_0811_7.jpeg" alt="图片名称" /></p> |
<p>Then we did gel extraction,the parameters of product are as follows:</p> | <p>Then we did gel extraction,the parameters of product are as follows:</p> | ||
<table class="ui celled table"> | <table class="ui celled table"> | ||
Line 1,057: | Line 1,057: | ||
<p>30 cycles(Duration ~ Extend)</p> | <p>30 cycles(Duration ~ Extend)</p> | ||
<p>Agarose gel electrophoresis Results: | <p>Agarose gel electrophoresis Results: | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/b/b8/T--USTC--NB_0811_8.jpeg" alt="图片名称" /> |
Agarose gel electrophoresis of PCR product</p> | Agarose gel electrophoresis of PCR product</p> | ||
<p>Failed again and again and again.</p> | <p>Failed again and again and again.</p> | ||
Line 1,352: | Line 1,352: | ||
<table class="ui celled table"> | <table class="ui celled table"> | ||
<p>After extraction, we conducted electrolysis to see if we got the desired product, the result of which is shown below. | <p>After extraction, we conducted electrolysis to see if we got the desired product, the result of which is shown below. | ||
− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2016/thumb/1/18/T--USTC--notebook_%281%29-22001.jpg/796px-T--USTC--notebook_%281%29-22001.jpg" /> |
From left to right, the stripes are corresponding to marker marker 2K, 1003, 1004, 1607, 1608 and marker 2K+ and the control plasmid.</p> | From left to right, the stripes are corresponding to marker marker 2K, 1003, 1004, 1607, 1608 and marker 2K+ and the control plasmid.</p> | ||
<p><strong>Gel Extraction for sfGFP11</strong> | <p><strong>Gel Extraction for sfGFP11</strong> | ||
Line 1,482: | Line 1,482: | ||
<table class="ui celled table"> | <table class="ui celled table"> | ||
<p>After enzymatic digestion, we conducted electrolysis to see if we got the desired product, the result of which is shown below. | <p>After enzymatic digestion, we conducted electrolysis to see if we got the desired product, the result of which is shown below. | ||
− | <img src=" | + | <img src="" /> |
This shows that we have got the desired outcome, sfGFP11. </p> | This shows that we have got the desired outcome, sfGFP11. </p> | ||
<p><strong>Gel Extraction for pro+RBS</strong> | <p><strong>Gel Extraction for pro+RBS</strong> |
Revision as of 17:42, 19 October 2016
Notebook
Together we stand
Performers
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DATE 8.12
Recorder: Yinchenguang Lyu & Yu Xie PCR of bacteria which have recombinant plasmid pYeT containing sfGFP1-10
Procedure:
- Prepare 4 PCR tubes and sequentially add:
sample | 1,2,3,4 | |||||||
---|---|---|---|---|---|---|---|---|
Sterilized ddH2O | 15.75 μL | |||||||
5×PrimeSTAR Buffer (Mg2+Plus) | 5 μL | |||||||
dNTP Mixture (2.5 mM each) | 2 μL | |||||||
Template(bacteria) | 1 μL | |||||||
Primer 1 | 0.5 μL | |||||||
Primer 2 | 0.5 μL | |||||||
PrimeSTAR HS DNA Polymerase (2.5 U/μl) | 0.25 μL | |||||||
total | 25 μL |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
Pre-Duration | 95 | 10 min | ||||||
Duration | 95 | 10 s | ||||||
Anneal | 55 | 15 s | ||||||
Extend | 72 | 44 s | ||||||
Post-Extend | 72 | 10 min | ||||||
Final | 4 | -- |
sample | 1,2,3,4 | |||||||
---|---|---|---|---|---|---|---|---|
Sterilized ddH2O | 15.75 μL | |||||||
5×PrimeSTAR Buffer (Mg2+Plus) | 5 μL | |||||||
dNTP Mixture (2.5 mM each) | 2 μL | |||||||
Template(bacteria) | 1 μL | |||||||
Primer 1 | 0.5 μL | |||||||
Primer 2 | 0.5 μL | |||||||
PrimeSTAR HS DNA Polymerase (2.5 U/μl) | 0.25 μL | |||||||
total | 25 μL |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
Pre-Duration | 95 | 10 min | ||||||
Duration | 95 | 10 s | ||||||
Anneal | 55 | 15 s | ||||||
Extend | 72 | 44 s | ||||||
Post-Extend | 72 | 10 min | ||||||
Final | 4 | -- |
sample | 1 | 2 | 3 | 4 | 5 | 6 | ||
---|---|---|---|---|---|---|---|---|
SterilizedddH2O | 11 μL | 11 μL | 11 μL | 11 μL | 11 μL | 11 μL | ||
2×HiFi-PCR Master | 12.5 μL | 0 μL | 12.5 μL | 0 μL | 12.5 μL | 0 μL | ||
PrimeSTAR premix | 0 μL | 12.5 μL | 0 μL | 12.5 μL | 0 μL | 12.5 μL | ||
Template (AD) | 0.5 μL | 0.5 μL | 0 μL | 0 μL | 0 μL | 0 μL | ||
Template (BD) | 0 μL | 0 μL | 0.5 μL | 0.5 μL | 0 μL | 0 μL | ||
Template (PFP) | 0 μL | 0 μL | 0 μL | 0 μL | 0.5 μL | 0.5 μL | ||
standard-biobrick-p | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | ||
standard-biobrick-r | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | 0.5 μL | ||
total | 25 μL | 25 μL | 25 μL | 25 μL | 25 μL | 25 μL |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
step 1 | 95 | 5 min | ||||||
step 2 | 95 | 1 min | ||||||
step 3 | 58 | 1 min | ||||||
step 4 | 72 | 45 s | ||||||
step 5 | 72 | 10 min | ||||||
step 6 | 4 | -- |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
step 1 | 95 | 5 min | ||||||
step 2 | 95 | 10 s | ||||||
step 3 | 55 | 15 s | ||||||
step 4 | 72 | 29 s | ||||||
step 5 | 72 | 10 min | ||||||
step 6 | 4 | -- |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
step 1 | 95 | 5 min | ||||||
step 2 | 95 | 1 min | ||||||
step 3 | 55 | 1 min | ||||||
step 4 | 72 | 2 min 8 s | ||||||
step 5 | 72 | 10 min | ||||||
step 6 | 4 | -- |
stage | temperature | time | ||||||
---|---|---|---|---|---|---|---|---|
step 1 | 95 | 5 min | ||||||
step 2 | 95 | 10 s | ||||||
step 3 | 55 | 15 s | ||||||
step 4 | 72 | 1 min 11 s | ||||||
step 5 | 72 | 10 min | ||||||
step 6 | 4 | -- |
Number | AD | BD | PFP | |||||
---|---|---|---|---|---|---|---|---|
A260/A280 | 1.85 | 1.87 | 1.91 | |||||
A260/A230 | 0.60 | 1.41 | 0.02 | |||||
concentration(ng/μL) | 69.9 | 73.9 | 86.7 |
Number | 0601 | 0602 | 0603 | 0604 | 1601 | 1602 | 1603 | 1604 |
---|---|---|---|---|---|---|---|---|
Content | sfGFP11 | sfGFP11 | sup35 | sup35 | sfGFP11 | sfGFP11 | sfGFP11 | sfGFP11 |
A260/A280 | 1.88 | 1.88 | 1.90 | 1.90 | 1.83 | 1.89 | 1.87 | 1.89 |
concentration(ng/μL) | 535.9 | 236.0 | 60.3 | 17.8 | 230.9 | 199.7 | 663.1 | 722.3 |
Number | 1001 | 1002 | 1003 | 1004 |
---|---|---|---|---|
Content | BD-PYW | AD-PYW | BD-PYW | AD-PYW |
A260/A280 | 2.02 | 1.79 | 1.79 | 1.98 |
concentration(ng/μL) | 359.7 | 115.8 | 124.0 | 87.1 |
Number | 1 | 2 | 3 | 4 |
---|---|---|---|---|
A260/A280 | 1.81 | 1.87 | 1.79 | 1.80 |
A260/A230 | 1.08 | 1.17 | 1.07 | 0.80 |
concentration(ng/μL) | 29.0 | 33.0 | 63.8 | 18.3 |
Number | 1 | 2 | 3 | 4 |
---|---|---|---|---|
A260/A280 | 1.89 | 1.88 | 1.88 | 1.88 |
A260/A230 | 2.24 | 2.18 | 2.12 | 2.17 |
concentration(ng/μL) | 416.6 | 398.5 | 382.9 | 429.4 |
Number | 1 | 2 | 3 | 4 |
---|---|---|---|---|
A260/A280 | 1.90 | 1.67 | 1.98 | 1.97 |
A260/A230 | 0.94 | 0.71 | 1.00 | 0.99 |
concentration(ng/μL) | 13.1 | 25.5 | 9.5 | 11.1 |
sample | 1,2,3,4,5,6,7,8 | |||
---|---|---|---|---|
Sterilized ddH2O | 9 μL | |||
2×HiFi-PCR Master | 12.5 μL | |||
Template(bacteria) | 1.5 μL | |||
Primer 1 | 1 μL | |||
Primer 2 | 1 μL | |||
total | 25 μL |
stage | temperature | time | ||
---|---|---|---|---|
Pre-Duration | 95 | 10 min | ||
Duration | 95 | 1 min | ||
Anneal | 58 | 1 min | ||
Extend | 72 | 1 min | ||
Post-Extend | 72 | 10 min | ||
Final | 4 | -- |
sample | 1,2,3,4,5,6,7,8 | |||
---|---|---|---|---|
Sterilized ddH2O | 9 μL | |||
2×HiFi-PCR Master | 12.5 μL | |||
Template(bacteria) | 1.5 μL | |||
Primer 1 | 1 μL | |||
Primer 2 | 1 μL | |||
total | 25 μL |
stage | temperature | time | ||
---|---|---|---|---|
Pre-Duration | 95 | 15 min | ||
Duration | 95 | 50s | ||
Anneal | 55 | 1 min | ||
Extend | 72 | 1min20s | ||
Post-Extend | 72 | 10 min | ||
Final | 4 | -- |
sample | 1,2,3,4,5,6,7,8 | |||
---|---|---|---|---|
Sterilized ddH2O | 9 μL | |||
2×HiFi-PCR Master | 12.5 μL | |||
Template(bacteria) | 1.5 μL | |||
Primer 1 | 1 μL | |||
Primer 2 | 1 μL | |||
total | 25 μL |
stage | temperature | time | ||
---|---|---|---|---|
Pre-Duration | 95 | 15 min | ||
Duration | 95 | 50s | ||
Anneal | 55 | 1 min | ||
Extend | 72 | 1min20s | ||
Post-Extend | 72 | 10 min | ||
Final | 4 | -- |
sample | 1 | 2 | 3 | 4 |
---|---|---|---|---|
A260/A280 | 1.87 | 1.88 | 1.88 | 1.87 |
A260/A230 | 1.49 | 1.23 | 2.12 | 2.07 |
concentration(ng/μL) | 143.2 | 190.8 | 546.2 | 459.8 |
sample | 1609 | 1010 | 1607 | 1608 |
---|---|---|---|---|
Content | pro+RBS | pro+RBS | pro+RBS | pro+RBS |
A260/A280 | 1.84 | 1.85 | 1.82 | 1.85 |
concentration(ng/μL) | 100.3 | 93.6 | 83.0 | 104.8 |
sample | 1003 | 1004 | 1607 | 1608 |
---|---|---|---|---|
Content | sup35+sfGFP11 | sup35+sfGFP11 | sup35+sfGFP11 | sup35+sfGFP11 |
A260/A280 | 1.86 | 1.86 | 1.87 | 1.87 |
concentration(ng/μL) | 229.7 | 235.5 | 233.4 | 241.7 |
sample | 1605 | 1606 |
---|---|---|
Content | sfGFP11 | sfGFP11 |
A260/A280 | 1.75 | 1.86 |
concentration(ng/μL) | 10.3 | 7.5 |
sample | 1511 | 1512 |
---|---|---|
Content | sfGFP11 | sfGFP11 |
A260/A280 | 1.86 | 1.86 |
concentration(ng/μL) | 215.4 | 152.8 |
sample | volume |
---|---|
Pstl | 2ul |
Xbal | 0.35ul |
10x DNA buffer | 2ul |
PCR product | 10 or 9ul |
ddH2O | 15.65 or 16.65ul |
total | 30ul |
sample | volume |
---|---|
Pstl | 1ul |
Xbal | 1ul |
10x DNA buffer | 2ul |
PCR product | 1ul |
total | 20ul |
sample | 1401 |
---|---|
Content | pro+RBS |
A260/A280 | 1.93 |
concentration(ng/μL) | 18.7 |