Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"
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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" style="text-indent:0em;"><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" style="text-indent:0em;"><li>β-mercaptoethanol</li></div></p> |
| − | <p><div class="text_font"><li>RNaseA</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>RNaseA</li></div></p> |
| − | <p><div class="text_font"><li>Chloroform</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Chloroform</li></div></p> |
| − | <p><div class="text_font"><li>Ice box and ice</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Ice box and ice</li></div></p> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 135: | Line 135: | ||
<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" style="text-indent:0em;"><li>ddH20</li></div></p> |
| − | <p><div class="text_font"><li>dNTPs mix</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>dNTPs mix</li></div></p> |
| − | <p><div class="text_font"><li>10×Buffer</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>10×Buffer</li></div></p> |
| − | <p><div class="text_font"><li>Taq polymerase</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Taq polymerase</li></div></p> |
| − | <p><div class="text_font"><li>PCR Primers</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>PCR Primers</li></div></p> |
| − | <p><div class="text_font"><li>Arabidopsis genomic DNA</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><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> | ||
<ol> | <ol> | ||
| − | <p><div class="text_font"><li> | + | <p><div class="text_font" style="text-indent:0em;"><li> |
add material into a 0.2ml EP tube according to the table</div></p> | add material into a 0.2ml EP tube according to the table</div></p> | ||
<table class="table"> | <table class="table"> | ||
| Line 185: | Line 185: | ||
</table> | </table> | ||
</li> | </li> | ||
| − | <p><div class="text_font"><li> | + | <p><div class="text_font" style="text-indent:0em;"><li> |
set the PCR program </div></p> | set the PCR program </div></p> | ||
<ul> | <ul> | ||
| Line 254: | Line 254: | ||
<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" style="text-indent:0em;"><li>ddH20</li></div></p> |
| − | <p><div class="text_font"><li>BsaⅠBuffer</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>BsaⅠBuffer</li></div></p> |
| − | <p><div class="text_font"><li>PstⅠBuffer</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>PstⅠBuffer</li></div></p> |
| − | <p><div class="text_font"><li>restriction enzyme BsaⅠ，PstⅠ，EcorⅠ</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><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" style="text-indent:0em;"><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> | + | <p><div class="text_font" style="text-indent:0em;"><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> | ||
<ol> | <ol> | ||
| − | <p><div class="text_font"><li> | + | <p><div class="text_font" style="text-indent:0em;"><li> |
add materials into a 1.5ml EP tube according to the table below:</div></p> | add materials into a 1.5ml EP tube according to the table below:</div></p> | ||
<table class="table"> | <table class="table"> | ||
| Line 338: | Line 338: | ||
</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" style="text-indent:0em;"><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> | + | <p><div class="text_font" style="text-indent:0em;"><li>Water bath the tube in 65℃ for 20 mins to end the digestion reaction.</li></div></p> |
<ol> | <ol> | ||
</div> | </div> | ||
| Line 351: | Line 351: | ||
<p><div class="text_font">(1)preparation of LB Broth</div></p> | <p><div class="text_font">(1)preparation of LB Broth</div></p> | ||
<ul> | <ul> | ||
| − | <p><div class="text_font"><li>2g peptone</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>2g peptone</li></div></p> |
| − | <p><div class="text_font"><li>1g NaCL</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>1g NaCL</li></div></p> |
| − | <p><div class="text_font"><li>0.2g glucose</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>0.2g glucose</li></div></p> |
| − | <p><div class="text_font"><li>1g yeast</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>1g yeast</li></div></p> |
| − | <p><div class="text_font"><li>Add water to 200ml, regulate the pH at a range of 6.8~7.2</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Add water to 200ml, regulate the pH at a range of 6.8~7.2</li></div></p> |
</ul> | </ul> | ||
<p><div class="text_font">Add water to 200ml, regulate the pH at a range of 6.8~7.2</div></p> | <p><div class="text_font">Add water to 200ml, regulate the pH at a range of 6.8~7.2</div></p> | ||
| Line 361: | Line 361: | ||
<p><div class="text_font">(3)Preparation of chloromycetin solution</div></p> | <p><div class="text_font">(3)Preparation of chloromycetin solution</div></p> | ||
<ul> | <ul> | ||
| − | <p><div class="text_font"><li>10ml ddH20</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>10ml ddH20</li></div></p> |
| − | <p><div class="text_font"><li>0.25g Chloromycetin</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>0.25g Chloromycetin</li></div></p> |
</ul> | </ul> | ||
<p><div class="text_font">Add trace amount of NaOH until the chloromycetin dissolves completely</div></p> | <p><div class="text_font">Add trace amount of NaOH until the chloromycetin dissolves completely</div></p> | ||
| Line 410: | Line 410: | ||
<p><div class="text_font">(1)preparation for RT-PCR</div></p> | <p><div class="text_font">(1)preparation for RT-PCR</div></p> | ||
<ul> | <ul> | ||
| − | <p><div class="text_font"><li>Buffer 5μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Buffer 5μl</li></div></p> |
| − | <p><div class="text_font"><li>Forward Primer 2μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Forward Primer 2μl</li></div></p> |
| − | <p><div class="text_font"><li>Reverse primer 2μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Reverse primer 2μl</li></div></p> |
| − | <p><div class="text_font"><li>RT reverse transcription 0.5μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>RT reverse transcription 0.5μl</li></div></p> |
| − | <p><div class="text_font"><li>Taq enzyme 0.5μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>Taq enzyme 0.5μl</li></div></p> |
| − | <p><div class="text_font"><li>RNA 20μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>RNA 20μl</li></div></p> |
| − | <p><div class="text_font"><li>ddH2O 19.5μl</li></div></p> | + | <p><div class="text_font" style="text-indent:0em;"><li>ddH2O 19.5μl</li></div></p> |
</ul> | </ul> | ||
<p><div class="text_font">(2)RT-PCR program</div></p> | <p><div class="text_font">(2)RT-PCR program</div></p> | ||
Revision as of 14:45, 16 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
- 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
| 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 |
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
- add materials into a 1.5ml EP tube according to the table below:
- 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.
| 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 |
4.chemical conversion
(1)preparation of LB Broth
- 2g peptone
- 1g NaCL
- 0.2g glucose
- 1g yeast
- Add water to 200ml, regulate the pH at a range of 6.8~7.2
Add water to 200ml, regulate the pH at a range of 6.8~7.2
(2)preparation of LB Broth Medium
(3)Preparation of chloromycetin solution
- 10ml ddH20
- 0.25g Chloromycetin
Add trace amount of NaOH until the chloromycetin dissolves completely
(4)add chloromycetin solution into LB broth, mix them
(5)Add competence DH5α （100μl）into 10 μl plasmid，mix them gently, and ice bath
(6)Heat the solution in 42℃ water bath，and ice-bath 2min (Be sure not to shake the centrifuge tube)
(7)Add 890μl, 37℃ preheating LB broth, mix them gently ,shake them in the condition 37℃, 150rpm for 45min.
(8)4000rpm centrifuge 5min, discard 900 μl supernatant ,suspend the 100μl sediment gentlely
(9)Spread the solution on the LB broth medium evenly, after it is dried, culture it upside down in the 37 ℃ incubator for 16 hours
(10)Put the centrifuge tube which including culture solution and competence DH5α in the bed temperature incubator, shaking culture it in the condition of 220rpm, 37℃ for 16 hours
*THE RESUALT：
(1)The escherichia coli didn't grow on the LB broth medium
(2)After centrifuged, some escherichia colis can be found with a little amount in the bottom of the centrifuge tubes
5.plasmid extraction
Experiment macterial: the kit for plasmid extraction
Experiment apparatus: centrifugal machine, water bath, incubator
Experiment preparation: 60 ℃ water bath， 37 ℃ incubator
(1)centrifuge the bacterial liqiud ,12000rpm 1min. Discard the supernatant.
(2)According to the kit for extraction, extract the plasmid DNA
(3)Preserve the plasmid in 4℃ refrigerator
6.extraction of RNA
Experiment macterial：the kit for RNA fractionation and purification
Experiment steps: refer to protocol
7.RT-PCR
Experiment macterial：M-MuLV
(1)preparation for RT-PCR
- Buffer 5μl
- Forward Primer 2μl
- Reverse primer 2μl
- RT reverse transcription 0.5μl
- Taq enzyme 0.5μl
- RNA 20μl
- ddH2O 19.5μl
(2)RT-PCR program
45℃×30min+94℃×5min+（94℃×30s+60.5℃×30s+72℃×1min）×40+72℃×10min
