Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"
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<div class="timeline"> | <div class="timeline"> | ||
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<div class="row"> | <div class="row"> | ||
| − | <div class=" | + | <div class="text-center"> |
| − | <h1>Protocol</h1> | + | <h1><strong>Protocol</strong></h1> |
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<p><div class="text_font">Material:</div></p> | <p><div class="text_font">Material:</div></p> | ||
<ul> | <ul> | ||
| − | + | <p><div class="text_font"><li>EZ-10 Spin Column Plant Genomic DNA Purification Kit</li></div></p> | |
| − | + | <p><div class="text_font"><li>β-mercaptoethanol</li></div></p> | |
| − | + | <p><div class="text_font"><li>RNaseA</li></div></p> | |
| − | + | <p><div class="text_font"><li>Chloroform</li></div></p> | |
| − | + | <p><div class="text_font"><li>Ice box and ice</li></div></p> | |
</ul> | </ul> | ||
</div> | </div> | ||
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<p><div class="text_font">Material:</div></p> | <p><div class="text_font">Material:</div></p> | ||
<ul> | <ul> | ||
| − | + | <p><div class="text_font"><li>ddH20</li></div></p> | |
| − | + | <p><div class="text_font"><li>dNTPs mix</li></div></p> | |
| − | + | <p><div class="text_font"><li>10×Buffer</li></div></p> | |
| − | + | <p><div class="text_font"><li>Taq polymerase</li></div></p> | |
| − | + | <p><div class="text_font"><li>PCR Primers</li></div></p> | |
| − | + | <p><div class="text_font"><li>Arabidopsis genomic DNA</li></div></p> | |
</ul> | </ul> | ||
<p><div class="text_font">Methods:</div></p> | <p><div class="text_font">Methods:</div></p> | ||
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<p><div class="text_font">Material:</div></p> | <p><div class="text_font">Material:</div></p> | ||
<ul> | <ul> | ||
| − | + | <p><div class="text_font"><li>ddH20</li></div></p> | |
| − | + | <p><div class="text_font"><li>BsaⅠBuffer</li></div></p> | |
| − | + | <p><div class="text_font"><li>PstⅠBuffer</li></div></p> | |
| − | + | <p><div class="text_font"><li>restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</li></div></p> | |
| − | + | <p><div class="text_font"><li>PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9</li></div></p> | |
| − | + | <p><div class="text_font"><li>Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9</li></div></p> | |
</ul> | </ul> | ||
<p><div class="text_font">Method</div></p> | <p><div class="text_font">Method</div></p> | ||
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</table> | </table> | ||
</li> | </li> | ||
| − | + | <p><div class="text_font"><li>Place the tube in a 37℃ incubator, stand for 1 hour.</li></div></p> | |
| − | + | <p><div class="text_font"><li>Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</li></div></p> | |
<ol> | <ol> | ||
</div> | </div> | ||
Revision as of 17:12, 1 October 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.
