Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"
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<div class="col-xs-3 date"></div> | <div class="col-xs-3 date"></div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | <div class="m-b-xs text_font"><strong>1.Extract Arabidopsis genomic DNA</strong></div> | + | <p><div class="m-b-xs text_font"><strong>1.Extract Arabidopsis genomic DNA</strong></div></p> |
| − | <div class="text_font">Material:</div> | + | <p><div class="text_font">Material:</div></p> |
<ul> | <ul> | ||
| − | <li><div class="text_font">EZ-10 Spin Column Plant Genomic DNA Purification Kit</div></li> | + | <li><p><div class="text_font">EZ-10 Spin Column Plant Genomic DNA Purification Kit</div></p></li> |
| − | <li><div class="text_font">β-mercaptoethanol</div></li> | + | <li><p><div class="text_font">β-mercaptoethanol</div></p></li> |
| − | <li><div class="text_font">RNaseA</div></li> | + | <li><p><div class="text_font">RNaseA</div></p></li> |
| − | <li><div class="text_font">Chloroform</div></li> | + | <li><p><div class="text_font">Chloroform</div></p></li> |
| − | <li><div class="text_font">Ice box and ice</div></li> | + | <li><p><div class="text_font">Ice box and ice</div></p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | <div class="m-b-xs text_font"><strong>2.PCR</strong></div> | + | <p><div class="m-b-xs text_font"><strong>2.PCR</strong></div></p> |
| − | <div class="text_font">Material:</div> | + | <p><div class="text_font">Material:</div></p> |
<ul> | <ul> | ||
| − | <li><div class="text_font">ddH20</div></li> | + | <li><p><div class="text_font">ddH20</div></p></li> |
| − | <li><div class="text_font">dNTPs mix</div></li> | + | <li><p><div class="text_font">dNTPs mix</div></p></li> |
| − | <li><div class="text_font">10×Buffer</div></li> | + | <li><p><div class="text_font">10×Buffer</div></p></li> |
| − | <li><div class="text_font">Taq polymerase</div></li> | + | <li><p><div class="text_font">Taq polymerase</div></p></li> |
| − | <li><div class="text_font">PCR Primers</div></li> | + | <li><p><div class="text_font">PCR Primers</div></p></li> |
| − | <li><div class="text_font">Arabidopsis genomic DNA</div></li> | + | <li><p><div class="text_font">Arabidopsis genomic DNA</div></p></li> |
</ul> | </ul> | ||
| − | <div class="text_font">Methods:</div> | + | <p><div class="text_font">Methods:</div></p> |
<ol> | <ol> | ||
<li> | <li> | ||
| − | <div class="text_font">add material into a 0.2ml EP tube according to the table</div> | + | <p><div class="text_font">add material into a 0.2ml EP tube according to the table</div></p> |
<table class="table"> | <table class="table"> | ||
<thead> | <thead> | ||
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</li> | </li> | ||
<li> | <li> | ||
| − | <div class="text_font">set the PCR program </div> | + | <p><div class="text_font">set the PCR program </div></p> |
<ul> | <ul> | ||
<li> | <li> | ||
| − | <div class="text_font">For promoter cop1,phyB,pif1,gene hhl1,gene flu:</div> | + | <p><div class="text_font">For promoter cop1,phyB,pif1,gene hhl1,gene flu:</div></p> |
| − | <div class="text_font">94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)</div> | + | <p><div class="text_font">94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)</div></p> |
</li> | </li> | ||
<li> | <li> | ||
| − | <div class="text_font">For pHhl1-gene hhl1:</div> | + | <p><div class="text_font">For pHhl1-gene hhl1:</div></p> |
| − | <div class="text_font">94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)</div> | + | <p><div class="text_font">94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)</div></p> |
</li> | </li> | ||
<li> | <li> | ||
| − | <div class="text_font">Tm of each primer: </div> | + | <p><div class="text_font">Tm of each primer: </div></p> |
<table class="table"> | <table class="table"> | ||
<thead> | <thead> | ||
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</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | <div class="m-b-xs text_font"><strong>3.digestion</strong></div> | + | <p><div class="m-b-xs text_font"><strong>3.digestion</strong></div></p> |
| − | <div class="text_font">Material:</div> | + | <p><div class="text_font">Material:</div></p> |
<ul> | <ul> | ||
| − | <li><div class="text_font">ddH20</div></li> | + | <li><p><div class="text_font">ddH20</div></p></li> |
| − | <li><div class="text_font">BsaⅠBuffer</div></li> | + | <li><p><div class="text_font">BsaⅠBuffer</div></p></li> |
| − | <li><div class="text_font">PstⅠBuffer</div></li> | + | <li><p><div class="text_font">PstⅠBuffer</div></p></li> |
| − | <li><div class="text_font">restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</div></li> | + | <li><p><div class="text_font">restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</div></p></li> |
| − | <li><div class="text_font">PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</div></li> | + | <li><p><div class="text_font">PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</div></p></li> |
| − | <li><div class="text_font">Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</div></li> | + | <li><p><div class="text_font">Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</div></p></li> |
</ul> | </ul> | ||
| − | <div class="text_font">Method</div> | + | <p><div class="text_font">Method</div></p> |
<ol> | <ol> | ||
<li> | <li> | ||
| − | <div class="text_font">1.add materials into a 1.5ml EP tube according to the table below:</div> | + | <p><div class="text_font">1.add materials into a 1.5ml EP tube according to the table below:</div></p> |
<table class="table"> | <table class="table"> | ||
<thead> | <thead> | ||
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</table> | </table> | ||
</li> | </li> | ||
| − | <li><div class="text_font">Place the tube in a 37℃ incubator, stand for 1 hour.</div></li> | + | <li><p><div class="text_font">Place the tube in a 37℃ incubator, stand for 1 hour.</div></p></li> |
| − | <li><div class="text_font">Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</div></li> | + | <li><p><div class="text_font">Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</div></p></li> |
<ol> | <ol> | ||
</div> | </div> | ||
Revision as of 13:21, 30 September 2016
Protocol
1.Extract Arabidopsis genomic DNA
Material:
- EZ-10 Spin Column Plant Genomic DNA Purification Kit
- β-mercaptoethanol
- RNaseA
- Chloroform
- Ice box and ice
2.PCR
Material:
- ddH20
- dNTPs mix
- 10×Buffer
- Taq polymerase
- PCR Primers
- Arabidopsis genomic DNA
Methods:
-
add material into a 0.2ml EP tube according to the table
Material dosage/μl dNTPs mix 1 Forward primer 0.5 Reverse primer 0.5 Arabidopsis genomic DNA 3 ddH20 12 Taq polymerase 0.5 Total 20 -
set the PCR program
-
For promoter cop1,phyB,pif1,gene hhl1,gene flu:94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)
-
For pHhl1-gene hhl1:94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)
-
Tm of each primer:
Name Tm(Forward/Reverse)(℃) Tm in PCR(℃) Promoter pif1 62.59/62.67 63 Promoter cop1 66.77/65.46 66 Promoter phyB 63.94/63.90 64 pHhl1-genehhl1 60.75/59.38 60 gene hhl1 60.90/59.38 60 Fluorescent gene flu 62.42/66.90 65
-
3.digestion
Material:
- ddH20
- BsaⅠBuffer
- PstⅠBuffer
- restriction enzyme BsaⅠ，PstⅠ，EcorⅠ
- PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9
- Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9
Method
-
1.add materials into a 1.5ml EP tube according to the table below:
Material dosage/μl PCR product(after purification) 17 restriction enzyme buffer 3 (/4) restriction enzyme 1(0.5 each) ddH20 4 Total 25
PCR product cutting sites restriction enzyme Buffer pHhl1-hhl1 BsaⅠ+EcorⅠ 3μlPstⅠ+1μlBsaⅠ hhl1 PstⅠ+EcorⅠ 3μlPstⅠ flu EcorⅠ+BsaⅠ 3μlPstⅠ+1μlBsa1Ⅰ cop1 BsaⅠ+PstⅠ 3μlPstⅠ phyB BsaⅠ+PstⅠ 3μlPstⅠ pif1 BsaⅠ+PstⅠ 3μlPstⅠ - Place the tube in a 37℃ incubator, stand for 1 hour.
- Water bath the tube in 65℃ for 20 mins to end the digestion reaction.
