发信人: bigfatliar (smurf), 信区: Biology
标 题: Re: Did I get correct cDNA clone?
发信站: The unknown SPACE (Tue Nov 5 15:35:17 2002) WWW-POST

【 在 chenligong (LaoChen) 的大作中提到: 】
: Can anyone give a brief steps about making a cDNA clone of a special gene?
how
: can you prove that that clone is what you want ?

1.extract the total mRNA
2.use restriction enzyme to cut out the desired gene
3.design a primer and do a PCR
4.Sequence the DNA and compare with those published sequences to see whether
it encodes the kind of enzyme you want.
5.Southern Blot to see whether pure and whether it is there.
6.Express the cDNA in Ecoli and check protein activity.
7.translate the sequenced cDNA to see whether you got the correct protein.
8.Sequence the expressed protein and back translate part of the AA's that has
the least codes to DNA sequence. Blast the sequence to check whether it
belongs to the group of gene that you are interested in.
9.Antibody for the specific expressed protein.Protein should be pure. Use
SDS-PAGE to check purity.


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