NGS DNA Library Purification via Dynabeads
Hello, I could use some help from the community.
I have built 48 RAD-Seq libraries using a protocol from Tin et al. (2014) designed for use with museum specimens. I have confirmed that the libraries have been built correctly via vector cloning of a positive control. After amplification using Phusion HF DNA Polymerase (40 cycles), all samples have been run on a gel to confirm that bands are produced around ~200 bp.
Then, I purified the PCR products via Dynabeads MyOne Carboxylic Aid as recommended by Tin et al. (2014). The protocol is as follows:
1. 10 µl per sample are removed from the bottle. The storage buffer is removed using a magnetic stand. Then, the beads are washed twice in EB and resuspended in EB maintaining the same volume.
2. 10 µl beads, 40 µl PCR product, and 100 µl of 14.5% PEG-6000 (0.9 M NaCl, 10 mM Tris, pH 6) are added.
3. The solution is incubation at room temperature for 10 minutes. The supernatant is removed using a magnetic stand and discarded. The beads are washed twice with 70% ethanol and air dried at room temperature.
4. Beads are eluted in 15 µl EB and incubated for 5 minutes at room temperature. The supernatant is kept.
To quantify, I ran a SYBER I Green Assay on all samples and got values between 0.2 ng/µl to 2 ng/µl. Next, I ran some samples of varying quantity (but within the range of the chip’s capabilities) on a Bioanalyzer High Sensitivity Chip. Typically, I rarely get a distribution, and if I do, the concentration is very small. Other times, I am getting unusual distributions that do not seem to have a pattern.
I have tried reamplifying for an additional 20 cycles and purifying again with no luck. I even replaced Phusion with GoTaq, which gave me unusual distributions. I am not sure what to make of this and believe something is wrong with the purification step. Does anyone have any experience with these beads or know of a solution?
I have contacted the manufacturers of the beads (Life Technologies), and they suggest high volume of beads and longer incubation times. I have already tried the longer incubation times with a positive control without any luck.
Thank you in advance.
I have built 48 RAD-Seq libraries using a protocol from Tin et al. (2014) designed for use with museum specimens. I have confirmed that the libraries have been built correctly via vector cloning of a positive control. After amplification using Phusion HF DNA Polymerase (40 cycles), all samples have been run on a gel to confirm that bands are produced around ~200 bp.
Then, I purified the PCR products via Dynabeads MyOne Carboxylic Aid as recommended by Tin et al. (2014). The protocol is as follows:
1. 10 µl per sample are removed from the bottle. The storage buffer is removed using a magnetic stand. Then, the beads are washed twice in EB and resuspended in EB maintaining the same volume.
2. 10 µl beads, 40 µl PCR product, and 100 µl of 14.5% PEG-6000 (0.9 M NaCl, 10 mM Tris, pH 6) are added.
3. The solution is incubation at room temperature for 10 minutes. The supernatant is removed using a magnetic stand and discarded. The beads are washed twice with 70% ethanol and air dried at room temperature.
4. Beads are eluted in 15 µl EB and incubated for 5 minutes at room temperature. The supernatant is kept.
To quantify, I ran a SYBER I Green Assay on all samples and got values between 0.2 ng/µl to 2 ng/µl. Next, I ran some samples of varying quantity (but within the range of the chip’s capabilities) on a Bioanalyzer High Sensitivity Chip. Typically, I rarely get a distribution, and if I do, the concentration is very small. Other times, I am getting unusual distributions that do not seem to have a pattern.
I have tried reamplifying for an additional 20 cycles and purifying again with no luck. I even replaced Phusion with GoTaq, which gave me unusual distributions. I am not sure what to make of this and believe something is wrong with the purification step. Does anyone have any experience with these beads or know of a solution?
I have contacted the manufacturers of the beads (Life Technologies), and they suggest high volume of beads and longer incubation times. I have already tried the longer incubation times with a positive control without any luck.
Thank you in advance.
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