FAQs and Tech Support

DNA Fragment Extraction- FAQs

Can PCR Advanced^TM Clean Up System be used to clean up DIG-labeled DNA fragment?

Yes, PX Buffer does not affect the chemically linked DIG on dNTP. Similarly, this system can be used to clean up ³²P-labeled DNA fragment.

What are the applications of PCR Advanced^TM Clean Up System? What is the difference between this system and gel extraction system?

Both systems can be used to clean up DNA fragments (100-bp to 10-kb) from enzymes, salts, and dNTPs. PCR Advanced^TM Clean Up System is a cheaper choice for cleaning up DNA fragments in solution. When a specific DNA fragment is to be purified, gel electrophoresis is needed. A gel slice containing the desired DNA fragment is excised, and DNA is extracted using Gel Advanced^TM Gel Extraction System.

Can PCR Advanced^TM Clean Up System be used to clean up dye terminator from sequencing reaction?

It is not recommended to use this system to clean up sequencing reaction because PCR products smaller than 100-bp cannot be recovered effectively, thus making reading of the first 80-100 nucleotide sequence unfeasible.

Why DNA recovery from an enzymatic reaction solution is lower than expected?

When the pH of the enzymatic reaction solution is higher than 7.5, DNA recovery will be reduced. In this case, add 10 µl of 3M potassium acetate of pH 5.0 to the DNA solution before adding PX Buffer.

Can this system completely remove primers or dimer products from PCR reactions?

PCR-M^® Clean Up System can only effectively (>90%) removes primers of less than 40-bp. When primers or dimer products are of more than 40-bp, they cannot be effectively removed. In this case, separate the PCR product from the dimer products by electrophoresis, excise the gel slice containing the desired product and purify it using Viogene Gel-M^® Gel Extraction System.

Why DNA recovery is lower than expected?

There are several possible reasons:

1) Do not overload the column with too much DNA. Higher recovery is attained when lower amount of DNA is loaded. Split loading high amount of DNA into more than one column.

2) If ddH2O is used for elution, make sure than its pH is between 7.0 and 8.5. pH lower than 7 leads to lower elution efficiency.

3) Make sure that complete DNA elution takes place by adding no less than 30 ml of elution solution onto the membrane and letting it completely absorbed into the membrane before centrifugation.

4) Large DNA fragment is eluted less readily than small DNA fragment. When the DNA product is larger than 5-kb, use elution solution preheated to 60˚C.

A band smaller than the desired DNA fragment is present in the eluant. What is this smaller band?

The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to that elongation of the PCR product is not complete or that PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA by incubating the solution at 95˚C for 2 minutes and let it cool slowly to room temperature. The re-annealed PCR product can be used as usual in all downstream applications.

How do I increase the recovery of the DNA fargment with small or large size?

Add 0.25 volume of isopropanol of the mixture from STEP 3. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp.

Add 0.25 volume of isopropanol of the mixture from STEP 1. and mix well. That will increase the recovery of the DNA, especially when the size of the DNA fragment is < 500-bp or > 5-kbp.

Tech Support

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