Hi, I tried 2 conditions on two different samples of different concentrations, and one sample gives an ultra concentration, I have an Idea of whats going on here, but I want more experimented opinions.
starting concentrations
1_ 1,08 ng/uL
2_ 2,57 ng/uL
I wanted to try if the ligation at Rt and at 20ºC makes some diference (20 minutes for all)
So I divided the samples taking 5uL each and diluting it to 5ng/uL with Elution Buffer, making 4 tubes, 2 tubes for sample so It ended up like this:
1.1
1.2
2.1
2.2
5ng/uL each
Then I went to end repair and A tailing, no problem at all.
On the adapter ligation I realized that on my tube of choice there was no volume for all samples (28nM final concentration) so I thaw a new adapter before using all the first one on 1.1, so I put the new adapter on 1.2, 2.1, 2.2.
1.1 and 2.1 went for adapter ligation for 20 minutes at Rt
1.2 and 2.2 went for adapter ligation on thermbloc at 20ºc for 20 minutes.
I´ve done the post adapter ligation cleanup with agencourt AMPure XP beads with no problem, I eluted the DNA with 20uL of elution buffer.
Then the samples went for library amplification for 12 cycles, and then I eluted the product again with the QIAquick MinElute kit columns & buffers.
Final concentrations goes like this (2uL samples on Qbit)
1.1- 3,32 ng/uL
1.2- 2,93 ng/uL
2.1- 6,96 ng/uL
2.2- 8.3 ng/uL
Is it possible that I didn´t resuspended well the new adapter after thawing it and after puting it in 1.2 so It was more concentrated and amplified more?
any other idea?
thank you so much!
You should have more DNA post amplification.
Additionally, ligation adds length to your sonicated DNA fragments. So assuming 100% bead recovery, the qubit would read “more” DNA.
Sample 2 also started with double quantity, so it make sense that amplified sample 2 would have higher DNA (remember amplification is exponential).
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column