Dynabeads™ DNA DIRECT™ Universal Kit

High PCR background could be caused by too high a concentration of template DNA, primers, Mg2+, or dNTPs. To fix this, reduce the amount of template DNA, primers, enzyme, and Mg2+ used, elute the DNA prior to PCR amplification, and/or try performing hot-start PCR. High background can also be caused by contamination. Ensure that the supernatant is completely removed at each washing step to avoid carryover.

We recommend eluting the DNA in Resuspension Buffer, water, or low ionic strength buffer by incubation at 65 degrees C for > 5 mins. The complex must be fully resuspended before elution. The elution buffer can be pre-heated to 65 degrees C. After incubation, place tube in magnetic stand for 30 secs and transfer supernatant to a clean tube. To determine the elution efficiency, you can run some of the eluted DNA on an agarose gel. Alternatively, you can perform a PCR with the Dynabeads magnetic beads to determine if there is residual DNA remaining on the beads. Using more pre-heated elution buffer would be helpful to improve the elution efficiency.

PCR may be inhibited by inhibitors, or an insufficient number of PCR cycles might have been used. For inhibitors, ensure that the washing buffer is brought to room temperature for use, and add the buffer more vigorously. You can also ensure that the supernatant is completely removed at each washing step, or introduce an additional washing step.

It might be possible to isolate bacterial DNA directly from soil but soil samples are difficult to work with, as there are a lot of PCR inhibitors present. Please check scientific literature for suggested protocols.

Lithium chloride is included in Washing Buffer A to ensure that the mRNA remains annealed to the Oligo(dT)25 on the beads while everything else is washed away. The major advantage of using LiCl instead of other chloride salts is that LiCl does not efficiently precipitate DNA, proteins, or carbohydrates and therefore reduces the risk of contamination of the final mRNA preparation with DNA and inhibitors of cDNA synthesis, PCR etc.

LiDS is an ionic detergent, similar in function to SDS. LiDS is included in the lysis buffer is to aid in the lysis of the cells and to denature proteins, and in addition it is an effective RNase inhibitor. If you don't have LiDS in the lab, it is also possible to use SDS, but you may wish to add RNAse inhibitor as well.