Difference between revisions of "GDSYZX-United/NOTEBOOK/day note"

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.

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.

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.

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.

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.

We use a kit box to purify our PCR products,so we can get our genes.

We did it.

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.

We purify our genes from the restriction enzyme systems.

We did it.

We cut our plasmids psb3c1 with BsaⅠ.

We did it.

We splice our DNA fragments to get the parts we want using a kit box.

We did it.

We purify our parts using a kit box.

We did it.

We cut our parts with BsaⅠto make sure there will be no loops in the part.

We did it.

We purify the parts.

We did it.

We splice our parts with plasmids.

We did it.

We prepare LB broth.

We did it.

We transform our plasmids into DH5α.

We did it.

We inoculate Ecoli DH5α on LB plates and in media.

We did it.

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.

We extract plasmids from the solution.

We did it.

We prepare Arabidopsis protoplasts.

The concentration of protoplasts is too low,this is because we chose the wrong filter paper.

We prepare protoplasts again.

We did it.The concentration is much higher,and there is much more sediment of protoplast after centrifuging.

We transfect our plasmids into the protoplasts，and incubate the protoplasts in the following light intensity (lux):0,3960,7920,11880.

We did it.

We extract RNA from protoplasts using a kit box.

We did it.

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.

PCR our target DNA fragments:cop1,pif1,phyB,hhl1,pHhl1-hhl1,flu.

We will check the result tomorrow.