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Old 11-01-2009, 03:40 PM   #1
seqgirl123
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Default A-base addition

In the polishing step for library preparation, there is a step using Klenow exo- polymerase and dATP, which will add one A-base to the 3' end of blunt ended DNA.

My question is how does only one A-base end up getting added onto the 3' end and not two or three?
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Old 01-16-2010, 07:52 PM   #2
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does anyone know the answer to this?
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Old 01-16-2010, 09:07 PM   #3
shurjo
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If recessed 3' ends are present, Klenow exo- fills them in and leaves a single base overhang in ~50% of cases. Given the blunt ends from the previous step in the Illumina protocol, it simply adds a single A to the 3' end.
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Old 01-17-2010, 02:33 AM   #4
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Thanks shurjo, that was very helpful!

Does it mean that, in the Illumina protocol, Klenow exo- adds a single A to ~50% of the blunted ends?
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Old 01-17-2010, 12:49 PM   #5
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What are the other 50% cases about?

Sometimes it will not add at all correct? Will it ever add 2 or more A' bases?
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