Difference between revisions of "GDSYZX-United/NOTEBOOK/day note"
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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>Extract genome from Arabidopsis Thaliana</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
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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>We use PCR to generate and to amplify our target DNA fragments:promotercop1,promoter phyB,promoterpif1,gene hhl1,gene pHhl,gene flu.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 158: | Line 158: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>Do the PCR again to get our target DNA fragments.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 174: | Line 174: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We use PCR to get cop1,phyB and pif1</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 189: | Line 189: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We use a kit box to purify our PCR products,so we can get our genes.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 204: | Line 204: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We cut our DNA fragments using restriction enzymes:BsaⅠ,PstⅠ, |
EcorⅠ.After this process ,the gene fragments will have one overhang at both ends.The overhangs will help them splice other fragments. </strong> | EcorⅠ.After this process ,the gene fragments will have one overhang at both ends.The overhangs will help them splice other fragments. </strong> | ||
</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>We purify our genes from the restriction enzyme systems.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
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<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We cut our plasmids psb3c1 with BsaⅠ.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 251: | Line 251: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We splice our DNA fragments to get the parts we want using |
a kit box.</strong> | a kit box.</strong> | ||
</div> | </div> | ||
| Line 267: | Line 267: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We purify our parts using a kit box.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 282: | Line 282: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We cut our parts with BsaⅠto |
make sure there will be no loops in the part.</strong> | make sure there will be no loops in the part.</strong> | ||
</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>We purify the parts.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 313: | Line 313: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We splice our parts with plasmids.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 328: | Line 328: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We prepare LB broth.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 343: | Line 343: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We transform our plasmids into DH5α.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 358: | Line 358: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We inoculate Ecoli DH5α on LB plates and in media.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 373: | Line 373: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We check the growth of Ecoli on LB plates.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 390: | Line 390: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We extract plasmids from the solution.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 405: | Line 405: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We prepare Arabidopsis protoplasts.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 420: | Line 420: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We prepare protoplasts again.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 435: | Line 435: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We transfect our plasmids into the protoplasts，and incubate |
the protoplasts in the following light intensity (lux):0,3960,7920,11880. | the protoplasts in the following light intensity (lux):0,3960,7920,11880. | ||
</strong> | </strong> | ||
| Line 452: | Line 452: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We extract RNA from protoplasts using a kit box.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
| Line 467: | Line 467: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>We use semiquantitive RT-PCR |
to amplify hhl1 to see how much hhl1 is transcripted into mRNA. | to amplify hhl1 to see how much hhl1 is transcripted into mRNA. | ||
</strong> | </strong> | ||
| Line 483: | Line 483: | ||
</div> | </div> | ||
<div class="col-xs-8 content"> | <div class="col-xs-8 content"> | ||
| − | < | + | <div class="m-b-xs text_font"><strong>PCR our target DNA fragments:cop1,pif1,phyB,hhl1,pHhl1-hhl1,flu.</strong> |
</div> | </div> | ||
<div class="text_font"> | <div class="text_font"> | ||
Revision as of 14:57, 29 September 2016
Day note
2016.7.31
9:00
Extract genome from Arabidopsis Thaliana
The genome has been degraded greatly,possibly because we didn’t grind the leaves in very low temperature to inhibit the nuclease.We have to do it again.
2016.7.31
13:30
Extract genome from Arabidopsis Thaliana
We extracted it the genome successfully.But the genome has still been degraded a little. (Since we don’t have liquid nitrogen in our lab,so we grinded the leaves in an ice box. )We will use the genome to do the following PCR.
2016.8.1
9:00
We use PCR to generate and to amplify our target DNA fragments:promotercop1,promoter phyB,promoterpif1,gene hhl1,gene pHhl,gene flu.
We failed to get all our target DNA fragments .We will check the primers and the PCR program ,and then do it again.
2016.8.2
9:00
Do the PCR again to get our target DNA fragments.
We PCR gene pHhl1,gene hhl1,gene flu successfully.
We will PCR cop1 together with pif1,phyB tomorrow.
2016.8.3
9:00
We use PCR to get cop1,phyB and pif1
In the first PCR we get cop 1 and pif1,we get higher concentration of phyB in the second time PCR.
2016.8.3
13:30
We use a kit box to purify our PCR products,so we can get our genes.
We did it.
2016.8.3
in the afternoon
We cut our DNA fragments using restriction enzymes:BsaⅠ,PstⅠ,
EcorⅠ.After this process ,the gene fragments will have one overhang at both ends.The overhangs will help them splice other fragments.
We did it.
2016.8.4
9:00
We purify our genes from the restriction enzyme systems.
We did it.
2016.8.4
in the morning
We cut our plasmids psb3c1 with BsaⅠ.
We did it.
2016.8.4
in the morning
We splice our DNA fragments to get the parts we want using
a kit box.
We did it.
2016.8.4
13:30
We purify our parts using a kit box.
We did it.
2016.8.4
in the afternoon
We cut our parts with BsaⅠto
make sure there will be no loops in the part.
We did it.
2016.8.4
in the afternoon
We purify the parts.
We did it.
2016.8.4
in the afternoon
We splice our parts with plasmids.
We did it.
2016.8.4
in the afternoon
We prepare LB broth.
We did it.
2016.8.4
19:00
We transform our plasmids into DH5α.
We did it.
2016.8.4
in the evening
We inoculate Ecoli DH5α on LB plates and in media.
We did it.
2016.8.5
13:00
We check the growth of Ecoli on LB plates.
No conlony was observed.
So we will use the Ecoli in media to do the following experiments.
2016.8.5
in the afternoon
We extract plasmids from the solution.
We did it.
2016.8.6
9:00
We prepare Arabidopsis protoplasts.
The concentration of protoplasts is too low,this is because we chose the wrong filter paper.
2016.8.6
13:00
We prepare protoplasts again.
We did it.The concentration is much higher,and there is much more sediment of protoplast after centrifuging.
2016.8.6
in the afternoon
We transfect our plasmids into the protoplasts，and incubate
the protoplasts in the following light intensity (lux):0,3960,7920,11880.
We did it.
2016.8.7
9:00
We extract RNA from protoplasts using a kit box.
We did it.
2016.8.7
in the morning
We use semiquantitive RT-PCR
to amplify hhl1 to see how much hhl1 is transcripted into mRNA.
We failed to get any hhl1. There may be something wrong with the process of transformation.
2016.8.10
PCR our target DNA fragments:cop1,pif1,phyB,hhl1,pHhl1-hhl1,flu.
We will check the result tomorrow.
