Hello everybody,
For a specific application, I have to sequence fragments of different sizes up to 900bp.
As a first step, I load my DNA sample onto a gel and cut the lane in five pieces, each piece representing a different size bin (in bp about 100-250/250-400/400-550/550-750/750-900).
The DNA is purified from each gel piece separately and used for library prep. We usually use the NEB Next DNA Library Prep kit adjusted for 10 ng of starting material.
In this case I use 10 ng of DNA for the 100-250 bp bin, 20 ng for the 250-400bp bin, etc to have about the same molar amounts of DNA in each reaction. Then I do normal blunting, A-tailing, and ligation of adapters.
After 9 cycles of PCR I measure the DNA concentration by QBit.
For small inserts, we aim at a final amount of about 500 ng. This I easily achieved for the two smallest bins (although in principal as the fragments become bigger, I would like to have proportionally more DNA in ng/ul to achieve similar molar amounts). The middle bin (400-550 bp) does also reach sufficient amounts after 2 more cycles. However, the two largest bins amplify very poorly. An initial problem was the amplification of self-ligated adapters. I seemed to get rid of those by purifying the ligation product twice after the ligation step with low AMPure/DNA ratios.
Now I see some amplified libraries for the two large bins, but only a small amount and after 4 additional cycles. I don't want to amplify too much further since this apparently can induce biases. Increasing primer conc. should not make a difference, and I also don't think that the nucleotides are limiting, since I am not reaching the amounts in ng/ul of the smaller fragments.
Could it be that the enzymatic reactions (blunting, A-tailing, adapter ligation) don't work as well on large fragments (although the concentrations of DNA ends should be constant among the different bins)?
Or do I have to adjust the PCR (I use Phusion polymerase and have adjusted the elongation time to 1 min).
Or does somebody have a protocol for library preps that works also for larger inserts (and is compatible with relatively low input amounts)?
Thank you very much!
Sandro
For a specific application, I have to sequence fragments of different sizes up to 900bp.
As a first step, I load my DNA sample onto a gel and cut the lane in five pieces, each piece representing a different size bin (in bp about 100-250/250-400/400-550/550-750/750-900).
The DNA is purified from each gel piece separately and used for library prep. We usually use the NEB Next DNA Library Prep kit adjusted for 10 ng of starting material.
In this case I use 10 ng of DNA for the 100-250 bp bin, 20 ng for the 250-400bp bin, etc to have about the same molar amounts of DNA in each reaction. Then I do normal blunting, A-tailing, and ligation of adapters.
After 9 cycles of PCR I measure the DNA concentration by QBit.
For small inserts, we aim at a final amount of about 500 ng. This I easily achieved for the two smallest bins (although in principal as the fragments become bigger, I would like to have proportionally more DNA in ng/ul to achieve similar molar amounts). The middle bin (400-550 bp) does also reach sufficient amounts after 2 more cycles. However, the two largest bins amplify very poorly. An initial problem was the amplification of self-ligated adapters. I seemed to get rid of those by purifying the ligation product twice after the ligation step with low AMPure/DNA ratios.
Now I see some amplified libraries for the two large bins, but only a small amount and after 4 additional cycles. I don't want to amplify too much further since this apparently can induce biases. Increasing primer conc. should not make a difference, and I also don't think that the nucleotides are limiting, since I am not reaching the amounts in ng/ul of the smaller fragments.
Could it be that the enzymatic reactions (blunting, A-tailing, adapter ligation) don't work as well on large fragments (although the concentrations of DNA ends should be constant among the different bins)?
Or do I have to adjust the PCR (I use Phusion polymerase and have adjusted the elongation time to 1 min).
Or does somebody have a protocol for library preps that works also for larger inserts (and is compatible with relatively low input amounts)?
Thank you very much!
Sandro
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