Question

What are the steps involved in cloning a gene?

Answer 1

If you want to clone the entire gene, it is more complicated. If you want to clone only the mRNA (for making an expression vector), it is simpler.

See Below

Explanation:

I'm going to assume you just want to make an expression vector of a mRNA - Lets say you want to clone the green fluorescent protein from your favorite Jelly Fish.

mRNA are the same sequence as the top strand of DNA, so a reverse primer for PCR can also function as a reverse transcriptase primer. Look up the GFP gene sequence and find its sequence. Use the last 20 nucleotides right up to the stop codon, and add a Sal1 restriction site to this primer. 5Sal1-gene specific sequence3.

Use this as your reverse transcriptase primer. Extract RNA from the poor jelly fish and then reverse transcribe the RNA using this primer.

Make a forward primer that is 5Sal1-ATG-1st Exon3' and then use the forward and reverse primer to amplify your cDNA library (which should have only GFP cDNA in it) by PCR. Now your cDNA for GFP has Sal1 sites at both ends. 5Sal1-ATG-cDNA-Stop-Sal1-3`

Take your favorite expression vector and cut it with Xho1 (assuming this is in your multiple cloning site.....oh yes, and you have to check that Sal1 and Xho1 are NOT in the cDNA sequence).

When the expression vector is cut with Xho1, add the cDNA and ligate. Xho1 and Sal1 are compatible sites, but when they form together, they kill both Xho1 and Sal1. After you have ligated the cDNA in, cut with Sal1 one last time (this will kill off the empty expression vectors that ligated back together and increase the number of correct plasmid/cDNA you will have for transformation.

Transform the plasmid into your favorite competent cells.

Use a forward primer in the last exon and a reverse primer on the 3` side of the multiple cloning site to colony screen your plate. Find your clone and grow it up.

After you've grown a bit of your clone, do a plasmid miniprep and seqence the multiple cloning site to ensure your cDNA is in place.

If you want to be able to cut the cDNA out, they clone into the Sal1 site on the multiple cloning site.

Now you've cloned your GFP protein cDNA and you can use it for whatever you like.