some words about how to improve your research ability
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When I read some posts, I often read some very basic questions. I always thinking, these questions are commoly taught by attending master or Ph.D courses. Or is easily solved by their tutors. Why they still ask such questions? So I write some of my thinkings, it may be benificial to new beginers. Due to my limited time, I can't explain in detail, and welcome whoever indicates if I made wrong.
Firstly, I want to say the basic technique we should to know.
1. cell culture. including sterilize, counting,passage,storage. The one thing I indicated here, you 'd better know the area of flask, dish, well , it very convenient for you to evaluate if the second day you need handling, it can save your time.
2. chemical and bio-molecule you are now handled, you should at least have basic understang of them. the most important thing is it's toxicity, and the fatality when you doing some work with isotope and irradiation and UV. Also the specilaties about protein,enzymes,antibodies,RNA,DNA,generally speaking, DNA is the most stable thing,
like plasmid, gonome DNA can be broken. RNA is vunebable, like primers,siRNA; protein is easily denatured and degraded so always on ice, to antibody and milk we need add some azide aginst bacteria.
3.we should know how to get the imformation from the net, I think the three tools are Pubmed,NCBI and google. e.g. how to get the cDNA sequence or protein domain ,MW from the net.
4.microscope, inverted , fluorenscence, cofocal microscope, the latter sometimes cause some difficulty, for it is too powerful with many functions.
5.Run agarose and PAGE gel, not needing say more except one thing,agarose gel is useful tool to purifying DNA.
6.Western blotting, it's very basic technique. Many persons ask questions about this, but it's easy. When for the first time to handle it, it's better use a known working antibody to a expessing stable protein,like CDK7,MCM7, never at the begining working with your own target protein. And I think, ponceau S staning is very important, from that, you know if you are suceesful boltted or not. Immunoprecipitation is based on WB.
7.For immunohistochemistry, cytostaining, FISH. it's easy to perform, but it's not easy to make a good staning under the microscope. So,for cytostaning, I want to both of acetone and formaldehylfe staining are OK, the former can be store for future use, the latter need permilization. And others, practice more, do more well.
8.PCR. 5 components: buffer,primer, template,dNTP,polymerase. Primer design is the determing factor commonly. If you run long fragments, I recommend pfu Turbo or Ultra. MSP or Site directed mutagenesis can cause some difficulties, it's not completely match. For the latter, it should be nitice, increase the concentration of template and reduce the cycles.
9. transfection. I use fugen6 for plasmid and Ologofectamine for siRNA,it 's effect simple. Calcium-Phosphate also works well, but sometimes I worry about it can cause some damage to the cell.
10.molecular cloning. I read a ph.D thesis in our country,it's main about constructing a plasmid. So i'm surprised. If works well, it commonly takes 3 days to construct a new plasmid. 1day, linealize the vector and run PCR for insert, 2nd purify and run on gel and ligation and transformation,3rd day make mini0prep and send sequencing. you need to sth. about T4 ligase ,about endonuclease. For PCR pruducts, my surgestion is overnight digestion,for restriction sites is oten at the end , it 's difficult to cut sometimes.
After all, I want to say, do experiments happily, if it's fail, it doesn't matter. Don't take it serious. Don't repeat one experiment again agian and it does not work, change another mehtod, every one can reach our goal. Sometimes if do the same experiment it always doesnot come out, it will make you frustrated, decrease your interest in scientific interest. Someone even cheating, in fact, it cheat yourself at last.
Finally, techniques are only tools to reach your goal, so the most important thing is
your research thought, so read more good articles, improve yourself.
Firstly, I want to say the basic technique we should to know.
1. cell culture. including sterilize, counting,passage,storage. The one thing I indicated here, you 'd better know the area of flask, dish, well , it very convenient for you to evaluate if the second day you need handling, it can save your time.
2. chemical and bio-molecule you are now handled, you should at least have basic understang of them. the most important thing is it's toxicity, and the fatality when you doing some work with isotope and irradiation and UV. Also the specilaties about protein,enzymes,antibodies,RNA,DNA,generally speaking, DNA is the most stable thing,
like plasmid, gonome DNA can be broken. RNA is vunebable, like primers,siRNA; protein is easily denatured and degraded so always on ice, to antibody and milk we need add some azide aginst bacteria.
3.we should know how to get the imformation from the net, I think the three tools are Pubmed,NCBI and google. e.g. how to get the cDNA sequence or protein domain ,MW from the net.
4.microscope, inverted , fluorenscence, cofocal microscope, the latter sometimes cause some difficulty, for it is too powerful with many functions.
5.Run agarose and PAGE gel, not needing say more except one thing,agarose gel is useful tool to purifying DNA.
6.Western blotting, it's very basic technique. Many persons ask questions about this, but it's easy. When for the first time to handle it, it's better use a known working antibody to a expessing stable protein,like CDK7,MCM7, never at the begining working with your own target protein. And I think, ponceau S staning is very important, from that, you know if you are suceesful boltted or not. Immunoprecipitation is based on WB.
7.For immunohistochemistry, cytostaining, FISH. it's easy to perform, but it's not easy to make a good staning under the microscope. So,for cytostaning, I want to both of acetone and formaldehylfe staining are OK, the former can be store for future use, the latter need permilization. And others, practice more, do more well.
8.PCR. 5 components: buffer,primer, template,dNTP,polymerase. Primer design is the determing factor commonly. If you run long fragments, I recommend pfu Turbo or Ultra. MSP or Site directed mutagenesis can cause some difficulties, it's not completely match. For the latter, it should be nitice, increase the concentration of template and reduce the cycles.
9. transfection. I use fugen6 for plasmid and Ologofectamine for siRNA,it 's effect simple. Calcium-Phosphate also works well, but sometimes I worry about it can cause some damage to the cell.
10.molecular cloning. I read a ph.D thesis in our country,it's main about constructing a plasmid. So i'm surprised. If works well, it commonly takes 3 days to construct a new plasmid. 1day, linealize the vector and run PCR for insert, 2nd purify and run on gel and ligation and transformation,3rd day make mini0prep and send sequencing. you need to sth. about T4 ligase ,about endonuclease. For PCR pruducts, my surgestion is overnight digestion,for restriction sites is oten at the end , it 's difficult to cut sometimes.
After all, I want to say, do experiments happily, if it's fail, it doesn't matter. Don't take it serious. Don't repeat one experiment again agian and it does not work, change another mehtod, every one can reach our goal. Sometimes if do the same experiment it always doesnot come out, it will make you frustrated, decrease your interest in scientific interest. Someone even cheating, in fact, it cheat yourself at last.
Finally, techniques are only tools to reach your goal, so the most important thing is
your research thought, so read more good articles, improve yourself.
