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In shotgun sequencing method or some related method that DNA is break up into random fragment. The fragment that have about 3kb in size is inserted into plasmid by enzyme ligase and then plasmid transform to E.coli using electroporator. As i understand, the goal is amplify the fragment to be sequencing.

At this time, i have a question :

  • After the e.coli growth up, we have too many different type of miscellaneous DNA strand with same size in a colony. How to purify a type of fragment to be sequencing ?

For example, i have 20.000 different DNA strand with the same 3kb in size after break up into random fragment :

-- AAAAAAAAAAAAAAAAAAAA...AAAAAAAAAAAAAAAAATTTTTTT -- (3kb fragment of chromosome 1)

-- GGGGXXXXXAAAAAAAAAAA...AAAAAAAAAAAAAAAAAAAAAAAA -- (3kb fragment of chromosome 2)

-- 19998 DNA fragment left ............................. -- (3kb fragment of random chromosome)

So after amplify them i have :

1000000x of -- AAAAAAAAAAAAAAAAAAAA...AAAAAAAAAAAAAAAAATTTTTTT --

1000000x of -- GGGGXXXXXAAAAAAAAAAA...AAAAAAAAAAAAAAAAAAAAAAAA --

1000000x of -- 19998 DNA fragment left........................ --

These DNA strand is mixed together in a colony. We cannot run sequencing them together as mixed, so how to have the only one for sequencing :

1000000x of -- AAAAAAAAAAAAAAAAAAAA...AAAAAAAAAAAAAAAAATTTTTTT --

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1 Answer 1

up vote 2 down vote accepted

I hope I understood your question correctly.

I think what you are missing is that after you break up the DNA, you dilute the concentration so that each bacteria will incorporate ~1 DNA molecule. Then, you dilute the bacteria on a plate and spread it so that they each single bacteria is far from other bacteria. Finally, after they grow you get one genetically identical colony per DNA fragment.

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You understood my question exactly and have a quick response, thank you so much ! –  Đức UltraSoft Jul 27 '13 at 4:57

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