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How to obtain the target gene

Update time : 2020-09-02

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How to obtain the target gene
The basic operating procedures of genetic engineering mainly include four steps: acquisition of target gene, construction of gene expression vector, introduction of target gene into recipient cells, detection and identification of target gene.
The following first introduces the acquisition of the target gene.

1. Determine the experiment object and experiment purpose.

For example, if we want to prepare genetically modified vegetables, we must choose a vegetable. Tomato, as the world's most productive vegetable, has a lot of related research. 

The genetic modification method is also mature. Everyone loves to eat it. It can be used as a vegetable and fruit. It is very convenient to grow at home and is a very good choice.

2. Find the target gene

Once we have determined the trait we want to change, then we need to find the gene that controls this trait.
Search for relevant gene sequence files on the Internet.

3. Gene cloning

Since we need to amplify a cDNA sequence from tomato species, this means that we have to extract tomato RNA, use reverse transcription to expand the cDNA, and then use DNA polymerase to expand the double-stranded gene fragment. 

The three main reagents here are reverse transcriptase, DNA polymerase (commonly known as PCR enzyme), and Trizol reagent for RNA extraction. PCR enzyme expansion of 2KB fragments with Taq enzyme is sufficient.

Then we can mention RNA! The extracted RNA is placed in the freezer.

Do you still remember the gene sequence file we downloaded earlier? Now we have to install some software to open it, design primers for gene amplification and construct vectors.

Then select the first 30-base positive-strand sequence and the last 30-base negative-strand sequence, and use them as primer sequences, and then find a biological company on the Internet that can synthesize primers, such as Generalbiosystems. 

Send them the sequence, and the primers will be sent to you in two days. The two primers are probably less than 7$. Then follow the instructions of the reverse transcriptase to perform reverse transcription with a PCR machine, and then use the reverse transcription product as a template to amplify the gene with the PCR machine according to the instructions of the PCR enzyme.

Then spend 10$ to buy a 5K DNA marker and agar powder for running the gel, and then send a dye (such as EB) that is added to the gel to stain the DNA, and run a gel to confirm that there is a 2K band. 

At this point, the gene cloning is complete. If you have spare money, you can send this PCR product to the company that orders the primers to test the sequence and confirm it.